Dithiol mucolytic agents

ABSTRACT

Provided are dithiol mucolytic agents. These agents increase the liquefaction of mucus in a patient with excessive mucus or mucus with increased viscoelastic, cohesive, or adhesive properties. Also provided are a variety of methods of treatment using these inventive mucolytic agents.

CONTINUING APPLICATION INFORMATION

This application claims benefit to U.S. provisional application Ser. No.61,869,378, filed on Aug. 23, 2013, and incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel dithiol mucolytic agents. Thepresent invention also includes a variety of methods of treatment usingthese inventive mucolytic agents.

2. Description of the Background

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. The mucus transport system is the fundamental defense of theairways against inhaled particulates/infectious agents. Inhaledparticles are trapped in the mucus layer and subsequently propelled outof the lungs via mucus clearance. The mucus transport system requiresthat mucus be well hydrated to facilitate cilliary clearance. In theabsence of sufficient mucus hydration, the mucus becomes excessivelyviscous and adherent, which can lead to airway mucus accumulation andinfection.

Typically, the quantity of the liquid layer on a mucosal surfacereflects the balance between epithelial liquid secretion, oftenreflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupled with water (and acation counter-ion), and epithelial liquid absorption, often reflectingNa⁺ absorption, coupled with water and counter anion (Cl⁻ and/or HCO₃⁻). Many diseases of mucosal surfaces are caused by too littleprotective liquid on those mucosal surfaces created by an imbalancebetween secretion (too little) and absorption (relatively too much). Thedefective salt transport processes that characterize these mucosaldysfunctions reside in the epithelial layer of the mucosal surface.

Abnormalities in the mucus transport system characterize a complex ofmuco-obstructive airway diseases that include cystic fibrosis (CF) andchronic bronchitis (CB). Normal mucus clearance requires 1) adequatehydration of the airway surface and 2) an absence of strong adhesive andcohesive interactions between mucus and cell surface. Hydration isdefined by the concentrations of mucins in the periciliary and mucuslayers. Ion transport properties regulate the amount of salt and water(i.e. the solvent) and goblet cells and glands control the quantity ofmucins on the airway surface. Subjects with mucus-obstructive diseasesincluding cystic fibrosis (CF), chronic bronchitis associated withcigarette smoke exposure, i.e., COPD, and asthma exhibit increases inmucus concentration as quantified by % solids (FIG. 1), as a result ofreduced airway hydration and mucin hypersecretion, consequent to gobletcell and glandular hyperplasia. Both as a function of disease severity,and in acute exacerbations, raised mucin concentrations produce adherentmucus that sticks to epithelial cells, impairs clearance, triggeringinflammatory responses and airway wall injury, and serves as a growthmedium for pathogenic microorganisms. Clearly, enhancing the clearanceof such thickened/adhered mucus from the airways is likely to benefitpatients with mucus-obstructive diseases.

Chronic bronchitis (CB), including the most common lethal genetic formof chronic bronchitis, cystic fibrosis (CF), are diseases that reflectthe body's failure to clear mucus normally from the lungs, whichultimately produces chronic airways infection. In the normal lung, theprimary defense against chronic intrapulmonary airways infection(chronic bronchitis) is mediated by the continuous clearance of mucusfrom bronchial airway surfaces. This function in health effectivelyremoves from the lung potentially noxious toxins and pathogens. Recentdata indicate that the initiating problem, i.e., the “basic defect,” inboth CB and CF is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance between the amount ofliquid and mucin on airway surfaces. This “airway surface liquid” (ASL)is primarily composed of salt and water in proportions similar to plasma(i.e., isotonic). Mucin macromolecules organize into a well-defined“mucus layer” which normally traps inhaled bacteria and is transportedout of the lung via the actions of cilia which beat in a watery, lowviscosity solution termed the “periciliary liquid” (PCL). In the diseasestate, there is an imbalance in the quantities of mucus and ASL onairway surfaces. This results in a relative reduction in ASL which leadsto mucus concentration, reduction in the lubricant activity of the PCL,and a failure to clear mucus via ciliary activity to the mouth. Thereduction in mechanical clearance of mucus from the lung leads tochronic bacterial colonization of mucus adherent to airway surfaces. Itis the chronic retention of bacteria, the failure of local antimicrobialsubstances to kill mucus-entrapped bacteria on a chronic basis, and theconsequent chronic inflammatory responses of the body to this type ofsurface infection, that lead to the syndromes of CB and CF.

The current afflicted population in the U.S. is 12,000,000 patients withthe acquired (primarily from cigarette smoke exposure) form of chronicbronchitis and approximately 30,000 patients with the genetic form,cystic fibrosis. Approximately equal numbers of both populations arepresent in Europe. In Asia, there is little CF but the incidence of CBis high and, like the rest of the world, is increasing.

There is currently a large, unmet medical need for products thatspecifically treat CB and CF at the level of the basic defect that causethese diseases. The current therapies for chronic bronchitis and cysticfibrosis focus on treating the symptoms and/or the late effects of thesediseases. Thus, for chronic bronchitis, β-agonists, inhaled steroids,anti-cholinergic agents, and oral theophyllines and phosphodiesteraseinhibitors are all in development. However, none of these drugs treateffectively the fundamental problem of the failure to clear mucus fromthe lung. Similarly, in cystic fibrosis, the same spectrum ofpharmacologic agents is used. These strategies have been complemented bymore recent strategies designed to clear the CF lung of the DNA(“Pulmozyme”; Genentech) that has been deposited in the lung byneutrophils that have futilely attempted to kill the bacteria that growin adherent mucus masses and through the use of inhaled antibiotics(“TOBI”) designed to augment the lungs' own killing mechanisms to ridthe adherent mucus plaques of bacteria. A general principle of the bodyis that if the initiating lesion is not treated, in this case mucusretention/obstruction, bacterial infections became chronic andincreasingly refractory to antimicrobial therapy. Thus, a major unmettherapeutic need for both CB and CF lung diseases is an effective meansof mobilizing airway mucus and promoting its clearance, with bacteria,from the lung.

Other mucosal surfaces in and on the body exhibit subtle differences inthe normal physiology of the protective surface liquids on theirsurfaces but the pathophysiology of disease reflects a common theme,i.e., too little protective surface liquid and impaired mucus clearance.For example, in xerostomia (dry mouth) the oral cavity is depleted ofliquid due to a failure of the parotid sublingual and submandibularglands to secrete liquid. Similarly, keratoconjunctivitis sicca (dryeye) is caused insufficient tear volume resulting from the failure oflacrimal glands to secrete liquid or excessive evaporative fluid loss.In rhinosinusitis, there is an imbalance, as in CB, between mucinsecretion, relative airway surface liquid depletion, and mucus stasis.Finally, in the gastrointestinal tract, failure to secrete Cl— (andliquid) in the proximal small intestine, combined with increased Na⁺(and liquid) absorption in the terminal ileum leads to the distalintestinal obstruction syndrome (DIOS). In older patients excessive Na⁺(and volume) absorption in the descending colon produces constipationand diverticulitis.

The high prevalence of both acute bronchitis and chronic bronchitisindicates that this disease syndrome is a major health problem in theU.S. Despite significant advancements in the etiology of mucusobstructive diseases, pharmacotherapy of both CF and COPD have beencharacterized by an aging array of therapies, typically includinginhaled steroids and bronchodilators for maintenance, and antibioticsand high-dose steroids for exacerbations. Clearly, what are needed aredrugs that are more effective at restoring the clearance of mucus fromthe lungs of patients with CB/CF. The value of these new therapies willbe reflected in improvements in the quality and duration of life forboth the CF and the CB populations.

One approach to increase mucus clearance is to enhance thetransportability of mucins via the disruption of the polymeric mucusstructure. Mucin proteins are organized into high molecular weightpolymers via the formation of covalent (disulfide) and non-covalentbonds. Disruption of the covalent bonds with reducing agents is awell-established method to reduce the viscoelastic properties of mucusin vitro and is predicted to minimize mucus adhesiveness and improveclearance in vivo. Reducing agents are well known to decrease mucusviscosity in vitro and commonly used as an aid to processing sputumsamples (Hirsch, S. R., Zastrow, J. E., and Kory, R. C. Sputumliquefying agents: a comparative in vitro evaluation. J. Lab. Clin. Med.1969. 74:346-353). Examples of reducing agents include sulfidecontaining molecules capable of reducing protein di-sulfide bondsincluding, but not limited to, N-acetyl cysteine, N-acystelyn,carbocysteine, cysteamine, glutathione, dithiothreitol (DTT), andthioredoxin containing proteins.

N-acetyl cysteine (NAC) is approved for use in conjunction with chestphysiotherapy to loosen viscid or thickened airway mucus. Clinicalstudies evaluating the effects of oral or inhaled NAC in CF and COPDhave reported improvements in the rheologic properties of mucus andtrends toward improvements in lung function and decreases in pulmonaryexacerbations (Duijvestijn Y C M and Brand P L P.; Systematic review ofN-acetylcysteine in cystic fibrosis. Acta Peadiatr 88: 38-41. 1999).However, the preponderance of clinical data suggests that NAC is at besta marginally effective therapeutic agent for treating airway mucusobstruction when administered orally or as an inhalation aerosol. Arecent Cochrane review of the existing clinical literature on the use ofNAC found no evidence to support the efficacy of NAC for CF (Nash E F,Stephenson A, Ratjen F, Tullis E.; Nebulized and oral thiol derivativesfor pulmonary disease in cystic fibrosis. Cochrane Database Syst Rev.2009; 21(1):CD007168.).

NAC, as a topical pulmonary therapeutic agent, is not optimal for thereduction of mucin disulfide bonds. Specifically, NAC does not possessthe basic properties of an effective pulmonary drug as NAC (1) is arelatively inefficient reducing agent the airway surface environment(e.g. CF pH 6.5-7.2); and (2) is rapidly metabolized and cleared fromthe airway surface (Jayaraman S. Song Y. Vetrivel L, Shankar L, VerkmanA S. Noninvasive in vivo fluorescence measurement of airway-surfaceliquid depth, salt concentration, and pH. J Clin Invest. 2001;107(3):317-24). For example, in the pH environment of the airway surface(measured in the range of pH 6.0 to 7.2 in CF and COPD airways), NACexists only partially in its reactive state as a negatively chargethiolate (Jayaraman S, Song Y, Vetrivel L, Shankar L, Verkman A S.Noninvasive in vivo fluorescence measurement of airway-surface liquiddepth, salt concentration, and pH. J Clin Invest. 2001; 107(3):317-24)(FIG. 3). Furthermore, in animal studies, ¹⁴C-labeled NAC, administeredby inhalation, exhibits rapid elimination from the lungs with ahalf-life of approximately 20 minutes (unpublished observation). Therelatively low reducing activity at of NAC physiologic airway pH and athe short half-life of NAC on the lung surface provide an explanationfor the lack of strong clinical evidence for effective mucus reductionin mucus obstructive diseases.

Additionally, NAC is most commonly administered as a concentratedinhalation solution (Mucomyst® is a 20% or 1.27M solution). However, theadministration of concentrated NAC solutions impact the tolerability ofNAC as it exaggerates (1) the unpleasant sulfur taste/odor; and (2)pulmonary side effects including irritation and bronchoconstrictionwhich can require co-administration of rescue medications such asbronchodilators. Although Mucomyst was approved by the FDA in 1963, noother reducing agents administered as an inhalation aerosol arecurrently available to treat muco-obstructive diseases. What are neededare effective, safe, and well-tolerated reducing agents for thetreatment of diseases characterized by impaired mucus clearance

SUMMARY OF THE INVENTION

One object of the present invention relates to a method to increase theliquefaction of mucus in a patient with excessive mucus or mucus withincreased viscoelastic, cohesive, or adhesive properties. The methodincludes the step of contacting the mucus of a patient with abnormal orexcessive mucus with a composition comprising a mucolytic compoundcontaining a dithiol group to decrease mucus viscoelasticity through thereduction of mucin disulfide bonds.

It is an object of the present invention to provide mucolytic compoundsthat are more effective, and/or absorbed less rapidly from mucosalsurfaces, and/or are better tolerated as compared to N-acetylcysteine(NAC) and DTT.

It is another object of the present invention to provide compounds whichare more active in the physiologic environment of the airway surface.

It is another object of the present invention to provide compounds thatare more potent and/or absorbed less rapidly, as compared to compoundssuch as N-acetylcysteine and DTT. Therefore, such compounds will give aprolonged pharmacodynamic half-life on mucosal surfaces as compared toNAC and DTT.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on promoting mucus clearance frommucosal surfaces.

It is an object of the present invention to provide compounds that aremore potent and/or absorbed less rapidly from mucosal surfaces, and/orare less reversible as compared to known compounds.

Therefore, the compounds will give a prolonged pharmacodynamic half-lifeon mucosal surfaces as compared to known compounds.

It is another object of the present invention to provide compounds whichare (1) absorbed less rapidly from mucosal surfaces, especially airwaysurfaces, as compared to known compounds and; (2) It is another objectof the present invention to provide compounds that are more potentand/or absorbed less rapidly and/or exhibit less reversibility, ascompared to compounds such as DTT and NAC. Therefore, such compoundswill give a prolonged pharmacodynamic half-life on mucosal surfaces ascompared to previous compounds.

It is another object of the present invention to provide methods oftreatment that take advantage of the pharmacological properties of thecompounds described above.

In particular, it is an object of the present invention to providemethods of treatment which rely on rehydration of mucosal surfaces.

The objects of the present invention may be accomplished with a class ofdithiols represented by compounds of Formula I which embraces structures(Ia)-(Id):

wherein R¹ and R² are each, independently, hydrogen, lower alkyl,halogen or triflouromethyl;

R³ and R⁴ are each, independently, hydrogen, lower alkyl, hydroxyl-loweralkyl, phenyl, (phenyl)-lower alkyl, (halophenyl)-lower alkyl,((lower-alkyl)phenyl)-lower-alkyl, ((lower-alkoxy)phenyl)-lower-alkyl,(naphthyl)-lower-alkyl, or (pyridyl)-lower-alkyl;

each R⁵ is, independently, hydrogen, halogen, trifluoromethyl, loweralkyl, unsubstituted or substituted phenyl, lower alkyl-thio,phenyl-lower alkyl-thio, lower alkyl-sulfonyl, or phenyl-loweralkyl-sulfonyl, OH, —(CH₂)_(n), —OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —(CH₂)_(n)—NR⁷R⁷,

—O—(CH₂)_(m)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R⁷,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —O—(CH₂),—(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸, —O—(CH₂CH₂O)_(n)—R⁸,—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—(CH₂)_(n)—C(═O)NR⁷R¹⁰, —O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m), —CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

-Link-(CH₂)_(m)-CAP, -Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CAP,-Link-(CH₂CH₂O)_(m)—CH₂—CAP, -Link-(CH₂CH₂O)_(m)—CH₂CH₂—CAP,-Link-(CH₂)_(m)—(Z)_(g)-CAP, -Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)—CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)—CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP, -Link-(CH₂)_(m)—(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—(CH₂)_(m), -CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)—CAP,-Link-(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)—(Z)_(g)-CAP, or-Link-Z_(g)—(CH₂)_(m)-Het-(CH₂)_(m)-CAP with the proviso that at leastone R⁵ group contains at least one basic nitrogen;

each R⁷ is, independently, hydrogen, lower alkyl, phenyl, substitutedphenyl, lower alkyl phenyl or —CH₂(CHOR⁸)_(m)—CH₂OR⁸;

each R⁸ is, independently, hydrogen, lower alkyl, lower alkyl phenyl,—C(═O)—R¹¹, glucuronide,

2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, —C(═O)R⁷,—CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or —C(═O)R¹³;

each R¹⁰ is, independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,or —CH₂—(CHOH)_(n)—CH₂OH;

each Z is, independently, —(CHOH)—, —C(═O)—, —(CHNR⁷R¹⁰°)—, —(C═NR¹⁰)—,—NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—, —(C═NR¹³)—, or —NR¹³—;

each R¹¹ is, independently, hydrogen, lower alkyl, phenyl lower alkyl orsubstituted phenyl lower alkyl;

each R¹² is, independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,—CH₂(CHOH)_(n)—CH₂OH, —CO₂R¹³, —C(═O)NR¹³R¹³, or —C(═O)R¹³;

each R¹³ is, independently, hydrogen, lower alkyl, phenyl, substitutedphenyl or —CH₂(CHOR⁸)_(m)—CH₂OR⁸, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹,—C(═O)R⁷, —CH₂—(CHOH)_(n)—CH₂OH, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(m)—NR⁷R⁷,—(CH₂)_(m)—NR¹¹R¹¹, —(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR¹¹R¹¹,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R¹⁰, —(CH₂)_(m)—NR¹⁰R¹⁰,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺,—(CH₂)_(m)—(CHOR⁸)_(m)(CH₂)_(m)NR⁷R⁷;

each g is, independently, an integer from 1 to 6; each m is,independently, an integer from 1 to 7;

each n is, independently, an integer from 0 to 7;

each -Het- is, independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or—CONR¹³—;

each Link is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m)—,—(CH₂)_(n)—(Z)_(g)—(CH₂)_(n)—, —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰—, or-Het-;

each CAP is, independently

with the proviso that when any —CHOR⁸— or —CH₂OR⁸ groups are located1,2- or 1,3- with respect to each other, the R⁸ groups may, optionally,be taken together to form a cyclic mono- or di-substituted 1,3-dioxaneor 1,3-dioxolane;

and racemates, enantiomers, diastereomers, tautomers, polymorphs,pseudopolymorphs and pharmaceutically acceptable salts, thereof.

The present invention also provides pharmaceutical compositions whichcomprise a compound as described herein.

The present invention also provides a method of restoring mucosaldefense, comprising: contacting mucus with an effective amount ofcompound described herein to a subject in need thereof.

The present invention also provides a method of decreasing mucusviscoelasticity, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method of decreasing mucusviscoelasticity on a mucosal surface, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method of scavenging free radicalson a mucosal surface, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method of decreasing inflammationon a mucosal surface, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method of reducing inflammatorycells on a mucosal surface, comprising:

administering an effective amount of a compound described herein to amucosal surface of a subject.

The present invention also provides a method treating mucus obstructivediseases, comprising:

contacting mucus with an effective amount of compound described hereinto a subject in need thereof.

The present invention also provides a method treating mucus adhesion,comprising:

contacting mucus with an effective amount of compound described hereinto a subject in need thereof.

The present invention also provides a method of treating chronicbronchitis, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating cysticfibrosis, comprising:

administering an effective amount of compound described herein to asubject in need thereof

The present invention also provides a method of treating cystic fibrosisexacerbations, comprising:

administering an effective amount of compound described herein to asubject in need thereof

The present invention also provides a method of treating bronchiectasis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating chronicobstructive pulmonary disease, comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating chronicobstructive pulmonary disease exacerbations, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating asthma,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating asthmaexacerbations, comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating esophagitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treatingventilator-induced pneumonia, comprising:

administering an effective compound described herein to a subject bymeans of a ventilator.

The present invention also provides a method of treating primary ciliarydyskinesia, comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating emphysema,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating pneumonia,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating rhinosinusitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating nasaldehydration, comprising:

administering an effective amount of a compound described herein to thenasal passages of a subject in need thereof

In a specific embodiment, the nasal dehydration is brought on byadministering dry oxygen to the subject.

The present invention also provides a method of treating sinusitis,comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating dry eye,comprising:

administering an effective amount of a compound described herein to theeye of the subject in need thereof.

The present invention also provides a method of promoting ocularhydration, comprising:

administering an effective amount of a compound described herein to theeye of the subject.

The present invention also provides a method of promoting cornealhydration, comprising:

administering an effective amount of a compound described herein to theeye of the subject.

The present invention also provides a method of treating excessive eyedischarge produced by, but not limited to blepharitis, allergies,conjunctivitis, corneal ulcer, trachoma, congenital herpes simplex,corneal abrasions, ectropion, eyelid disorders, gonococcalconjunctivitis, herpetic keratitis, ophthalmitis, Sjogren's Syndrome,Stevens-Johnson Syndrome comprising:

administering an effective amount of a compound described herein to theeye of the subject.

The present invention also provides a method of treating Sjögren'sdisease, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating dry mouth(xerostomia), comprising:

administering an effective amount of compound described herein to themouth of the subject in need thereof.

The present invention also provides a method of treating vaginaldryness, comprising:

administering an effective amount of a compound described herein to thevaginal tract of a subject in need thereof.

The present invention also provides a method of treating constipation,comprising:

administering an effective amount of a compound described herein to asubject in need thereof. In one embodiment of this method, the compoundis administered either orally or via a suppository or enema.

The present invention also provides a method of treating distalintestinal obstruction syndrome, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating chronicdiverticulitis comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of inducing sputum fordiagnostic purposes, comprising:

administering an effective amount of compound described herein to asubject in need thereof.

The present invention also provides a method of treating inhaledpathogens, comprising:

administering an effective amount of a compound described herein to asubject in need thereof

The present invention also provides a method of treating inhaledirritants, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

The present invention also provides a method of treating inhaledparticles, comprising:

administering an effective amount of a compound described herein to asubject in need thereof.

In a specific embodiment, the inhaled particles are insoluble particlesincluding dust, debris, or radioactive material.

The objects of the invention may also be accomplished with a method oftreating anthrax, comprising administering an effective amount of acompound of Formula I as defined herein and an osmolyte to a subject inneed thereof.

The objects of the invention may also be accomplished with a method ofprophylactic, post-exposure prophylactic, preventive or therapeutictreatment against diseases or conditions caused by pathogens,particularly pathogens which may be used in bioterrorism, comprisingadministering an effective amount of a compound of Formula I to asubject in need thereof.

It is further an object of the present invention to provide treatmentscomprising the use of osmolytes together with mucolytics of Formula Ithat are more potent, more specific, and/or absorbed less rapidly frommucosal surfaces as compared to compounds such as NAC.

It is another aspect of the present invention to provide treatmentsusing mucolytics of Formula I that are more potent and/or absorbed lessrapidly and/or exhibit less reversibility, as compared to compounds suchas NAC when administered with an osmotic enhancer. Therefore, suchmucolytics when used in conjunction with osmolytes will give anincreased pharmacodynamic effect on mucosal surfaces as compared toeither compound used alone.

It is another object of the present invention to provide treatmentsusing mucolytics of Formula I and osmolytes together which are absorbedless rapidly from mucosal surfaces, especially airway surfaces than NAC.It is another object of the invention to provide compositions whichcontain mucolytics of Formula I and osmolytes.

The objects of the invention may be accomplished with a method oftreating a disease ameliorated by increased mucus clearance and mucosalhydration comprising administering an effective amount of a compound ofFormula I as defined herein and an osmolyte to a subject in need ofincreased mucociliary clearance and/or mucosal hydration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Role of mucus dehydration in pathogenic sequence of CF/COPD.Disease-related dehydration of mucus (panel B, t % solids), leads to acollapse of the periciliary layer (PCL), reduction or cessation of mucusclearance, and adhesion of the mucus layer to the cell surface.

FIG. 2. Reduction of human salivary mucin by compounds L, N, and O.Human saliva was exposed to a range of concentrations of compounds L, Nand O or DTT, as indicated, electrophoresed, transferred to anitrocellulose membrane, and probed for Muc5b. Concentrations areindicated in mM and reduction reactions were performed for 30 min at 25°C. Reactions were quenched with NEM prior to electrophoresis.

FIG. 3. Reduction of human salivary mucin by compounds W and Y. Humansaliva was exposed to a range of concentrations of compounds W and Y, asindicated, electrophoresed, transferred to a nitrocellulose membrane,and probed for Muc5b. Concentrations are indicated in mM and reductionreactions were performed for 30 min at 25° C. Reactions were quenchedwith NEM prior to electrophoresis.

FIG. 4. Reduction of human salivary mucin by compounds H and G. Humansaliva was exposed to a range of concentrations of compounds H and G, asindicated, electrophoresed, transferred to a nitrocellulose membrane,and probed for Muc5b. Concentrations are indicated in mM and reductionreactions were performed for 30 min at 25° C. Reactions were quenchedwith NEM prior to electrophoresis.

FIG. 5. Reduction of human salivary mucin by compounds H and NAC. Humansaliva was exposed to 10 mM of compounds H or NAC, as indicated,electrophoresed, transferred to a nitrocellulose membrane, and probedfor Muc5b. Reduction reactions were performed for a range of times, asindicated in min., at 25° C. Reactions were quenched with NEM prior toelectrophoresis.

FIG. 6. Reduction of human salivary mucin by compounds KK, L, and O.Human saliva was exposed to 1 mM of compounds KK, L and O or DTT, asindicated, electrophoresed, transferred to a nitrocellulose membrane,and probed for Muc5b. Reduction reactions were performed for a range oftimes, as indicated in min., at 25° C. Reactions were quenched with NEMprior to electrophoresis.

FIG. 7. Reduction of human salivary mucin by compounds W and LL. Humansaliva was exposed to 0.3 mM (left panel) or 1 mM (right panel) ofcompounds W and LL, as indicated, electrophoresed, transferred to anitrocellulose membrane, and probed for Muc5b. Reduction reactions wereperformed for a range of times, as indicated in min., at 25° C.Reactions were quenched with NEM prior to electrophoresis.

FIG. 8. Tracheal Mucus Velocity in sheep for compound G.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms are defined as indicated.

“A compound of the invention” means a compound of Formula I or a salt,particularly a pharmaceutically acceptable salt thereof.

“A compound of Formula I” means a compound having the structural formuladesignated herein as Formula I. Compounds of Formula I include solvatesand hydrates (i.e., adducts of a compound of Formula I with a solvent).In those embodiments wherein a compound of Formula I includes one ormore chiral centers, the phrase is intended to encompass each individualstereoisomer including optical isomers (enantiomers and diastereomers)and geometric isomers (cis-/trans-isomerism) and mixtures ofstereoisomers. In addition, compounds of Formula I also includetautomers of the depicted formula(s).

Throughout the description and examples, compounds are named usingstandard IUPAC naming principles, where possible, including the use ofthe ChemDraw Ultra 11.0 software program for naming compounds, sold byCambridgeSoft Corp./PerkinElmer.

In some chemical structure representations where carbon atoms do nothave a sufficient number of attached variables depicted to produce avalence of four, the remaining carbon substituents needed to provide avalence of four should be assumed to be hydrogen. Similarly, in somechemical structures where a bond is drawn without specifying theterminal group, such bond is indicative of a methyl (Me, —CH₃) group, asis conventional in the art.

The present invention is based on the discovery that the compounds ofFormula I are more potent and/or, absorbed less rapidly, achieve higherconcentrations and have higher residence time in the mucosal surfaces,especially airway surfaces, and/or are better tolerated compared to NACand DTT. Therefore, the compounds of Formula I have a greater activityand/or produce less cellular toxicity on mucosal surfaces as compared toNAC and DTT.

The present invention is based on the discovery that the compounds offormula (I) are more potent and/or, absorbed less rapidly from mucosalsurfaces, especially airway surfaces, and/or less reversible frominteractions as compared to compounds such as NAC and DTT. Therefore,the compounds of formula (I) have a longer half-life on mucosal surfacesas compared to these compounds.

In the compounds represented by formula I which embraces structures(Ia)-(Id):

R¹ is independently hydrogen, lower alkylkoxy, halogen ortriflouromethyl;

R² is, independently hydrogen or lower alkyl;

R³ and R⁴ are each, independently, hydrogen, lower alkyl, hydroxyl-loweralkyl, phenyl, (phenyl)-lower alkyl, (halophenyl)-lower alkyl,((lower-alkyl)phenyl)-lower-alkyl, ((lower-alkoxy)phenyl)-lower-alkyl,(naphthyl)-lower-alkyl, or (pyridyl)-lower-alkyl;

each R⁵ is, independently, hydrogen, halogen, trifluoromethyl, loweralkyl, unsubstituted or substituted phenyl, lower alkyl-thio,phenyl-lower alkyl-thio, lower alkyl-sulfonyl, or phenyl-loweralkyl-sulfonyl, OH, —(CH₂)_(m)—OR^(B), —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —(CH₂)_(n)—NR⁷R⁷,

—O—(CH₂)_(m)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R⁷,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷, —O—(CH₂)_(m)(Z)_(g)—R⁷,—(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

-Link-(CH₂)_(m)—CAP, -Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂CH₂O)_(m)—CH₂—CAP, -Link-(CH₂CH₂O)_(m)—CH₂CH₂—CAP,-Link-(CH₂)_(m)—(Z)_(g)-CAP, -Link-(CH₂)—(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)—CAP, -Link-(CH₂)(CHOR⁸)_(m)CH₂—NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(m)—(Z)_(g)—(CH₂)_(m)—CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)—CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)—(Z)_(g)-CAP, or-Link-Z_(g)—(CH₂)_(m)—Het-(CH₂)_(m)—CAP with the proviso that at leastone R⁵ group contains at least one basic nitrogen;

The term —O-glucuronide, unless otherwise specified, means a grouprepresented by

wherein the

O means the glycosidic linkage can be above or below the plane of thering.

The term —O-glucose, unless otherwise specified, means a grouprepresented by

wherein the

O means the glycosidic linkage can be above or below the plane of thering.

In a preferred embodiment R⁵ is one of the following:

hydrogen, halogen, trifluoromethyl, lower alkyl, unsubstituted orsubstituted phenyl, lower alkyl-thio, phenyl-lower alkyl-thio, loweralkyl-sulfonyl, or phenyl-lower alkyl-sulfonyl, OH, —(CH₂)_(m)—OR⁸,—O—(CH₂)_(m)—OR⁸, —(CH₂)_(n)—NR⁷R¹⁰, —O—(CH₂)_(m)—NR⁷R¹⁰,—(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

In a preferred embodiment, each —(CH₂)_(n)—(Z)_(g)—R⁷ falls within thescope of the structures described above and is, independently,—(CH₂)_(n)—NH—C(═NH)NH₂,

In another a preferred embodiment, each —O—(CH₂)_(m)—(Z)_(g)—R⁷ fallswithin the scope of the structures described above and is,independently,—O—(CH₂)_(m)—NH—C(═NH)—N(R⁷)₂, or—O—(CH₂)_(m)—CHNH₂—CO₂NR⁷R¹⁰

In another preferred embodiment, R⁵ is —OH, —O—(CH₂)_(m)(Z)_(g)R¹²,-Het-(CH₂)_(m)—NH—C(═NR¹³)—NR¹³R¹³,-Het-(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)NH—C(═NR¹³)—NR¹³R¹³,-Link-(CH₂)_(m)—(Z)_(g)—(CH₂)_(m)-CAP, Link-(CH₂)_(n)—CR¹¹R¹¹-CAP,-Het-(CH₂)_(m)—CONR¹³R¹³, —(CH₂)_(n)—NR¹²R¹², —O—(CH₂)_(m)NR¹¹R¹¹,—O—(CH₂)_(m)—N^(⊕)—(R¹¹)₃, —(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)—NR¹⁰R¹⁰, -Het-(CH₂)_(m)—(Z)_(g)—NH—C(═NR¹³)—NR¹³R¹³,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —O—(CH₂)_(m)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—(Z)_(g)—R⁷, or—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸.

In a particularly preferred embodiment, R⁵ is -Link-(CH₂)_(m)—CAP,(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP, -Link-(CH₂CH₂O)_(m)—CH₂—CAP,-Link-(CH₂CH₂O)_(m)—CH₂CH₂—CAP, -Link-(CH₂)_(m)—(Z)_(g)—CAP,-Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m) (CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(m)—(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)—(Z)_(g)-CAP, or-Link-Z_(g)—(CH₂)_(m)-Het-(CH₂)_(m)-CAP.

Selected substituents within the compounds of the invention are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number ofcompounds may be present in any given embodiment. For example, R⁹contains a R¹³ substituent. R¹³ can contain an R¹⁰ substituent and R¹⁰can contain a R⁹ substituent. One of ordinary skill in the art ofmedicinal chemistry understands that the total number of suchsubstituents is reasonably limited by the desired properties of thecompound intended. Such properties include, by way of example and notlimitation, physical properties such as molecular weight, solubility orlog P, application properties such as activity against the intendedtarget, and practical properties such as ease of synthesis.

By way of example and not limitation, R⁵, R¹³ and R¹⁰ are recursivesubstituents in certain embodiments. Typically, each of these mayindependently occur 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2, 1, or 0, times in a given embodiment. More typically,each of these may independently occur 12 or fewer times in a givenembodiment. More typically yet, R⁹ will occur 0 to 8 times in a givenembodiment, R¹³ will occur 0 to 6 times in a given embodiment and R¹⁰will occur 0 to 6 times in a given embodiment. Even more typically yet,R⁹ will occur 0 to 6 times in a given embodiment, R¹³ will occur 0 to 4times in a given embodiment and R¹⁰ will occur 0 to 4 times in a givenembodiment.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal chemistry understands theversatility of such substituents. To the degree that recursivesubstituents are present in an embodiment of the invention, the totalnumber will be determined as set forth above.

Each -Het- is, independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂—; —O—,—SO₂NH—, —NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or—CONR¹³—. In a preferred embodiment, -Het- is —O—, —N(R⁷)—, or —N(R¹⁰)—.Most preferably, -Het- is —O—.

Each -Link- is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m) ⁻,—(CH₂)_(n)—(Z)_(g)—(CH₂)_(n) ⁻, —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰—,or -Het-. In a preferred embodiment, -Link- is —O—, —(CH₂)_(n)—,—NR¹³—C(═O)—(CH₂)_(m)—, or —C(═O)NR¹³—(CH₂)_(m) ⁻.

Each -CAP is each CAP is, independently

In a preferred embodiment, CAP is

Each g is, independently, an integer from 1 to 6. Therefore, each g maybe 1, 2, 3, 4, 5, or 6.

Each m is an integer from 1 to 7. Therefore, each m may be 1, 2, 3, 4,5, 6, or 7.

Each n is an integer from 0 to 7. Therefore, each n may be 0, 1, 2, 3,4, 5, 6, or 7.

Each Z is, independently, —(CHOH)—, —C(═O)—, —(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—,—NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—, —(C═NR¹³)—, or —NR¹³—. As designatedby (Z)_(g) in certain embodiments, Z may occur one, two, three, four,five or six times and each occurrence of Z is, independently, —(CHOH)—,—C(═O)—, —(CHNR⁷R¹⁰)—, —(C═NR¹⁰)—, —NR¹⁰—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—,—(C═NR¹³)—, or —NR¹³—. Therefore, by way of example and not by way oflimitation, (Z)_(g) can be —(CHOH)—(CHNR⁷R¹⁰)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—, —(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—(CHNR¹³R¹³)—,—(CHOH)—(CHNR⁷R¹⁰)—C(═O)—(CH₂)_(n)—(CHNR¹³R¹³)—C(═O)—, and the like.

In any variable containing —CHOR⁸— or —CH₂OR⁸ groups, when any —CHOR⁸—or —CH₂OR⁸ groups are located 1,2- or 1,3- with respect to each other,the R⁸ groups may, optionally, be taken together to form a cyclic mono-or di-substituted 1,3-dioxane or 1,3-dioxolane.

The compounds described herein may be prepared and used as the freebase. Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceuticallyacceptable salts of compounds within the scope of formulae I (Ia-Id) areembraced by the present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

A compound of formula I and its pharmaceutically acceptable salts mayexist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism means the ability of a hydrate or solvate of acompound to exist in different crystal structures. The pseudopolymorphsof the instant invention may exist due to differences in crystal packing(packing pseudopolymorphism) or due to differences in pakcing betweendifferent conformers of the same molecule (conformationalpseudopolymorphism). The instant invention comprises all polymorphs andpseudopolymorphs of the compounds of formula I-III and theirpharmaceutically acceptable salts.

A compound of formula I and its pharmaceutically acceptable salts mayalso exist as an amorphous solid. As used herein, an amorphous solid isa solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention comprises all amorphous forms of the compounds of formulaI-III and their pharmaceutically acceptable salts.

The compounds of formula I may exist in different tautomeric forms. Oneskilled in the art will recognize that amidines, amides, guanidines,ureas, thioureas, heterocycles and the like can exist in tautomericforms. All possible tautomeric forms of the amidines, amides,guanidines, ureas, thioureas, heterocycles and the like of all of theembodiments of formula I are within the scope of the instant invention.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and l, D and L, or (+) and (−) areemployed to designate the sign of rotation of plane-polarized light bythe compound, with S, (−), or l meaning that the compound islevorotatory while a compound prefixed with R, (+), or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

A single stereoisomer, e.g. an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

The compounds described herein may be prepared and used as the freebase. Alternatively, the compounds may be prepared and used as apharmaceutically acceptable salt. Pharmaceutically acceptable salts aresalts that retain or enhance the desired biological activity of theparent compound and do not impart undesired toxicological effects.Examples of such salts are (a) acid addition salts formed with inorganicacids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid,phosphoric acid, nitric acid and the like; (b) salts formed with organicacids such as, for example, acetic acid, oxalic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolicacid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid,phthalic acid, mandelic acid, lactic acid and the like; and (c) saltsformed from elemental anions for example, chlorine, bromine, and iodine.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceuticallyacceptable salts of compounds within the scope of formulae (X areembraced by the present invention. All mixtures of such enantiomers anddiastereomers are within the scope of the present invention.

A compound of formula I and its pharmaceutically acceptable salts mayexist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism means the ability of a hydrate or solvate of acompound to exist in different crystal structures. The pseudopolymorphsof the instant invention may exist due to differences in crystal packing(packing pseudopolymorphism) or due to differences in packing betweendifferent conformers of the same molecule (conformationalpseudopolymorphism). The instant invention comprises all polymorphs andpseudopolymorphs of the compounds of formula I-III and theirpharmaceutically acceptable salts.

A compound of formula I and its pharmaceutically acceptable salts mayalso exist as an amorphous solid. As used herein, an amorphous solid isa solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention comprises all amorphous forms of the compounds of formula Iand their pharmaceutically acceptable salts.

The compounds of formula I may exist in different tautomeric forms. Oneskilled in the art will recognize that amidines, amides, guanidines,ureas, thioureas, heterocycles and the like can exist in tautomericforms. All possible tautomeric forms of the amidines, amides,guanidines, ureas, thioureas, heterocycles and the like of all of theembodiments of formula I-IV are within the scope of the instantinvention.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and l, D and L, or (+) and (−) areemployed to designate the sign of rotation of plane-polarized light bythe compound, with S, (−), or l meaning that the compound islevorotatory while a compound prefixed with R, (+), or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

A single stereoisomer, e.g. an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. L.Eliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

As discussed above, the compounds used to prepare the compositions ofthe present invention may be in the form of a pharmaceuticallyacceptable free base. Because the free base of the compound is generallyless soluble in aqueous solutions than the salt, free base compositionsare employed to provide more sustained release of active agent to thelungs. An active agent present in the lungs in particulate form whichhas not dissolved into solution is not available to induce aphysiological response, but serves as a depot of bioavailable drug whichgradually dissolves into solution.

In a preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

In another preferred embodiment, the compound of formula (I) is

The present invention also provides methods of treatment that takeadvantage of the properties of the compounds described herein asdiscussed above. Thus, subjects that may be treated by the methods ofthe present invention include, but are not limited to, patientsafflicted with cystic fibrosis, asthma, primary ciliary dyskinesia,chronic bronchitis, bronchiectasis chronic obstructive airway disease,artificially ventilated patients, patients with acute pneumonia, etc.The present invention may be used to obtain a sputum sample from apatient by administering the active compounds to at least one lung of apatient, and then inducing or collecting a sputum sample from thatpatient. Typically, the invention will be administered to respiratorymucosal surfaces via aerosol (liquid or dry powders) or lavage.

Subjects that may be treated by the method of the present invention alsoinclude patients being administered supplemental oxygen nasally (aregimen that tends to dry the airway surfaces); patients afflicted withan allergic disease or response (e.g., an allergic response to pollen,dust, animal hair or particles, insects or insect particles, etc.) thataffects nasal airway surfaces; patients afflicted with a bacterialinfection e.g., staphylococcus infections such as Staphylococcus aureusinfections, Hemophilus influenza infections, Streptococcus pneumoniaeinfections, Pseudomonas aeuriginosa infections, etc.) of the nasalairway surfaces; patients afflicted with an inflammatory disease thataffects nasal airway surfaces; or patients afflicted with sinusitis(wherein the active agent or agents are administered to promote drainageof congested mucous secretions in the sinuses by administering an amounteffective to promote drainage of congested fluid in the sinuses), orcombined, Rhinosinusitis. The invention may be administered torhino-sinal surfaces by topical delivery, including aerosols and drops.

The present invention may be used to improve mucus clearance other thanairway surfaces. Such other mucosal surfaces include gastrointestinalsurfaces, oral surfaces, genito-urethral surfaces, and ocular surfacesor surfaces of the eye. For example, the active compounds of the presentinvention may be administered by any suitable means, includinglocally/topically, orally, or rectally, in an effective amount.

In another aspect, a post-exposure prophylactic treatment or therapeutictreatment method is provided for treating infection from an airbornepathogen comprising administering an effective amount of the compoundsof formula (I-VII) to the lungs of an individual in need of suchtreatment against infection from an airborne pathogen. The pathogenswhich may be protected against by the prophylactic post exposure, rescueand therapeutic treatment methods of the invention include any pathogenswhich may enter the body through the mouth, nose or nasal airways, thusproceeding into the lungs. Typically, the pathogens will be airbornepathogens, either naturally occurring or by aerosolization. Thepathogens may be naturally occurring or may have been introduced intothe environment intentionally after aerosolization or other method ofintroducing the pathogens into the environment. Many pathogens which arenot naturally transmitted in the air have been or may be aerosolized foruse in bioterrorism. The pathogens for which the treatment of theinvention may be useful includes, but is not limited to, category A, Band C priority pathogens as set forth by the NIAID. These categoriescorrespond generally to the lists compiled by the Centers for DiseaseControl and Prevention (CDC). As set up by the CDC, Category A agentsare those that can be easily disseminated or transmittedperson-to-person, cause high mortality, with potential for major publichealth impact. Category B agents are next in priority and include thosethat are moderately easy to disseminate and cause moderate morbidity andlow mortality. Category C consists of emerging pathogens that could beengineered for mass dissemination in the future because of theiravailability, ease of production and dissemination and potential forhigh morbidity and mortality. Particular examples of these pathogens areanthrax and plague. Additional pathogens which may be protected againstor the infection risk therefrom reduced include influenza viruses,rhinoviruses, adenoviruses and respiratory syncytial viruses, and thelike. A further pathogen which may be protected against is thecoronavirus which is believed to cause severe acute respiratory syndrome(SARS).

The present invention also relates to the use of mucolytic agents ofFormula I, or a pharmaceutically acceptable salt thereof, forpreventing, mitigating, and/or treating deterministic health effects tothe respiratory tract caused by exposure to radiological materials,particularly respirable aerosols containing radionuclides from nuclearattacks, such as detonation of radiological dispersal devices (RDD), oraccidents, such as nuclear power plant disasters. As such, providedherein is a method for preventing, mitigating, and/or treatingdeterministic health effects to the respiratory tract and/or otherbodily organs caused by respirable aerosols containing radionuclides ina recipient in need thereof, including in a human in need thereof, saidmethod comprising administering to said human an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

A major concern associated with consequence management planning forexposures of members of the public to respirable aerosols containingradionuclides from nuclear attacks, such as detonation of radiologicaldispersal devices (RDD), or accidents, such as nuclear power plantdisasters is how to prevent, mitigate or treat potential deterministichealth effects to the respiratory tract, primarily the lung. It isnecessary to have drugs, techniques and procedures, and trainedpersonnel prepared to manage and treat such highly internallycontaminated individuals.

Research has been conducted to determine ways in which to prevent,mitigate or treat potential damage to the respiratory tract and variousorgans in the body that is caused by internally deposited radionuclides.To date, most of the research attention has focused on strategiesdesigned to mitigate health effects from internally depositedradionuclides by accelerating their excretion or removal. Thesestrategies have focused on soluble chemical forms that are capable ofreaching the blood stream and are deposited at remote systemic sitesspecific to a given radioelement. Such approaches will not work in caseswhere the deposited radionuclide is in relatively insoluble form.Studies have shown that many, if not most of the physicochemical formsof dispersed radionuclides from RDDs, will be in relatively insolubleform.

The only method known to effectively reduce the radiation dose to thelungs from inhaled insoluble radioactive aerosols is bronchoalveolarlavage or BAL. This technique, which was adapted from that already inuse for the treatment of patients with alveolar proteinosis, has beenshown to be a safe, repeatable procedure, even when performed over anextended period of time. Although there are variations in procedure, thebasic method for BAL is to anaesthetize the subject, followed by theslow introduction of isotonic saline into a single lobe of the lunguntil the function residual capacity is reached. Additional volumes arethen added and drained by gravity.

The results of studies using BAL on animals indicate that about 40% ofthe deep lung content can be removed by a reasonable sequence of BALs.In some studies, there was considerable variability among animals in theamount of radionuclide recovered. The reasons for the variability arecurrently not understood.

Further, based on a study on animals, it is believed that a significantdose reduction from BAL therapy results in mitigation of health effectsdue to inhalation of insoluble radionuclides. In the study, adult dogsinhaled insoluble ¹⁴⁴Ce-FAP particles. Two groups of dogs were givenlung contents of ¹⁴⁴Ce known to cause radiation pneumonitis andpulmonary fibrosis (about 2 MBq/kg body mass), with one group beingtreated with 10 unilateral lavages between 2 and 56 days after exposure,the other untreated. A third group was exposed at a level of ¹⁴⁴Cecomparable to that seen in the BAL-treated group after treatment (about1 MBq/kg), but these animals were untreated. All animals were allowed tolive their lifespans, which extended to 16 years. Because there isvariability in initial lung content of ¹⁴⁴Ce among the dogs in eachgroup, the dose rates and cumulative doses for each group overlap.Nevertheless, the effect of BAL in reducing the risk frompneumonitis/fibrosis was evident from the survival curves. In theuntreated dogs with lung contents of 1.5-2.5 MBq/kg, the mean survivaltime was 370±65 d. For the treated dogs, the mean survival was 1270±240d, which was statistically significantly different. The third group,which received lung contents of ¹⁴⁴Ce of 0.6-1.4 MBq had a mean survivaltime of 1800±230, which was not statistically different from the treatedgroup. Equally important to the increased survival, the dogs in thehigh-dose untreated group died from deterministic effects to lung(pneumonitis/fibrosis) while the treated dogs did not. Instead, thetreated dogs, like the dogs in the low-dose untreated group, mostly hadlung tumors (hemangiosarcoma or carcinoma). Therefore, the reduction indose resulting from BAL treatment appears to have produced biologicaleffects in lung that were predictable based on the radiation doses thatthe lungs received.

Based on these results, it is believed that decreasing the residualradiological dose further by any method or combination of methods forenhancing the clearance of particles from the lung would furtherdecrease the probability of health effects to lung. However, BAL is aprocedure that has many drawbacks. BAL is a highly invasive procedurethat must be performed at specialized medical centers by trainedpulmonologists. As such, a BAL procedure is expensive. Given thedrawbacks of BAL, it is not a treatment option that would be readily andimmediately available to persons in need of accelerated removal ofradioactive particles, for example, in the event of a nuclear attack. Inthe event of a nuclear attack or a nuclear accident, immediate andrelatively easily administered treatment for persons who have beenexposed or who are at risk of being exposed is needed. Sodium channelblockers administered as an inhalation aerosol have been shown torestore hydration of airway surfaces. Such hydration of airway surfacesaids in clearing accumulated mucus secretions and associated particulatematter from the lung. As such, without being bound by any particulartheory, it is believed that sodium channel blockers can be used incombination with mucolytic agents described in this invention toaccelerate the removal of radioactive particles from airway passages.

As discussed above, the greatest risk to the lungs following aradiological attack, such as a dirty bomb, results from the inhalationand retention of insoluble radioactive particles. As a result ofradioactive particle retention, the cumulative exposure to the lung issignificantly increased, ultimately resulting in pulmonaryfibrosis/pneumonitis and potentially death. Insoluble particles cannotbe systemically cleared by chelating agents because these particles arenot in solution. To date, the physical removal of particulate matterthrough BAL is the only therapeutic regimen shown to be effective atmitigating radiation-induced lung disease. As discussed above, BAL isnot a realistic treatment solution for reducing the effects ofradioactive particles that have been inhaled into the body. As such, itis desirable to provide a therapeutic regimen that effectively aids inclearing radioactive particles from airway passages and that, unlikeBAL, is relatively simple to administer and scalable in a large-scaleradiation exposure scenario. In addition, it is also desirable that thetherapeutic regimen be readily available to a number of people in arelatively short period of time.

In an aspect of the present invention, a method for preventing,mitigating, and/or treating deterministic health effects to therespiratory tract and/or other bodily organs caused by respirableaerosols containing radionuclides comprises administering an effectiveamount of a mucolytic agent of Formula I or a pharmaceuticallyacceptable salt thereof to an individual in need. In a feature of thisaspect, the mucolytic agent is administered in conjunction with anosmolyte. With further regard to this feature, the osmolyte ishypertonic saline. In a further feature, the mucolytic agent and theosmolyte are administered in conjunction with an ion transportmodulator. With further regard to this feature, the ion transportmodulator may be selected from the group consisting of β-agonists, CFTRpotentiators, purinergic receptor agonists, lubiprostones, and proteaseinhibitors. In another feature of this aspect, the radionuclides areselected from the group consisting of Colbalt-60, Cesium-137,Iridium-192, Radium-226, Phospohrus-32, Strontium-89 and 90, Iodine-125,Thallium-201, Lead-210, Thorium-234, Uranium-238, Plutonium, Cobalt-58,Chromium-51, Americium, and Curium. In a further feature, theradionuclides are from a radioactive disposal device. In yet anotherfeature, the mucolytic agent or pharmaceutically acceptable salt thereofis administered in an aerosol suspension of respirable particles whichthe individual inhales. In an additional feature, the mucolytic agent ora pharmaceutically acceptable salt thereof is administered post-exposureto the radionuclides.

The present invention is concerned primarily with the treatment of humansubjects, but may also be employed for the treatment of other mammaliansubjects, such as dogs and cats, for veterinary purposes.

Another aspect of the present invention is a pharmaceutical composition,comprising a compound of formula I in a pharmaceutically acceptablecarrier (e.g., an aqueous carrier solution). In general, the compound offormula I is included in the composition in an amount effective toreduce the viscosity of mucus on mucosal surfaces.

An aspect of the present invention is the combination of mucolyticagents with other drugs or excipients to improve the efficacy andtolerability of the compounds described in the present invention.

Another aspect of the present invention is administering potent reducingagents in combination with osmolytes. A simple means to restore airwaysurface hydration in subjects with muco-obstructive diseases is toinhale hypertonic osmolyte solutions (most frequently 7% hypertonicsaline (HS)), which draws water onto the airway surface. Rehydration ofthe lubricant periciliary layer (PCL) of the airway surface facilitatesmucus clearance and, therefore, the removal of inhaled infectiousagents.

Inhaled HS is a unique therapeutic agent as it is used by ˜60% of CFpatients nationwide, but is not FDA approved for daily use for pulmonarydisease. As such, HS has not undergone the rigorous clinical testing toidentify the dose and dosing frequency that are most efficacious andwell tolerated. Instead, the HS regime has been optimized in practice bypatients and physicians. Most commonly, HS is administered as two 15minute inhalation treatments of 4 mL of 7% hypertonic saline pertreatment. The tonicity of HS used by patients (7% NaCl) has beenidentified as a maximum concentration that is generally tolerated (i.e.minimal irritation or bronchoconstriction).

Another approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Otherapproaches to hydrate the airway surface include chloride (Cl⁻)secretogogues that draw Cl⁻ and water into the ASL.

The compounds of Formula I may also be used in conjunction withosmolytes thus lowering the dose of the compound needed to hydratemucosal surfaces. This important property means that the compound willhave a lower tendency to cause undesired side-effects. Active osmolytesof the present invention are molecules or compounds that are osmoticallyactive (i.e., are “osmolytes”). “Osmotically active” compounds of thepresent invention are membrane-impermeable (i.e., essentiallynon-absorbable) on the airway or pulmonary epithelial surface. The terms“airway surface” and “pulmonary surface,” as used herein, includepulmonary airway surfaces such as the bronchi and bronchioles, alveolarsurfaces, and nasal and sinus surfaces. Active compounds of the presentinvention may be ionic osmolytes (i.e., salts), or may be non-ionicosmolytes (i.e., sugars, sugar alcohols, and organic osmolytes). It isspecifically intended that both racemic forms of the active compoundsthat are racemic in nature are included in the group of active compoundsthat are useful in the present invention. It is to be noted that allracemates, enantiomers, diastereomers, tautomers, polymorphs andpseudopolymorphs and racemic mixtures of the osmotically activecompounds are embraced by the present invention.

Active compounds of the present invention may be ionic osmolytes (i.e.,salts), or may be non-ionic osmolytes (i.e., sugars, sugar alcohols, andorganic osmolytes). It is specifically intended that both racemic formsof the active compounds that are racemic in nature are included in thegroup of active compounds that are useful in the present invention. Itis to be noted that all racemates, enantiomers, diastereomers,tautomers, polymorphs and pseudopolymorphs and racemic mixtures of theosmotically active compounds are embraced by the present invention.

Active osmolytes useful in the present invention that are ionicosmolytes include any salt of a pharmaceutically acceptable anion and apharmaceutically acceptable cation. Preferably, either (or both) of theanion and cation are non-absorbable (i.e., osmotically active and notsubject to rapid active transport) in relation to the airway surfaces towhich they are administered. Such compounds include but are not limitedto anions and cations that are contained in FDA approved commerciallymarketed salts, see, e.g., Remington: The Science and Practice ofPharmacy, Vol. II, pg. 1457 (19.sup.th Ed. 1995), incorporated herein byreference, and can be used in any combination including theirconventional combinations.

Pharmaceutically acceptable osmotically active anions that can be usedto carry out the present invention include, but are not limited to,acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide,calcium edetate, camsylate (camphorsulfonate), carbonate, chloride,citrate, dihydrochloride, edetate, edisylate (1,2-ethanedisulfonate),estolate (lauryl sulfate), esylate (1,2-ethanedisulfonate), fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate(p-glycollamidophenylarsonate), hexylresorcinate, hydrabamine(N,N-Di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride,hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate,maleate, mandelate, mesylate, methylbromide, methylnitrate,methylsulfate, mucate, napsylate, nitrate, nitrte, pamoate (embonate),pantothenate, phosphate or diphosphate, polygalacturonate, salicylate,stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate(8-chlorotheophyllinate), triethiodide, bicarbonate, etc. Particularlypreferred anions include chloride sulfate, nitrate, gluconate, iodide,bicarbonate, bromide, and phosphate.

Pharmaceutically acceptable cations that can be used to carry out thepresent invention include, but are not limited to, organic cations suchas benzathine (N,N′-dibenzylethylenediamine), chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine),procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium,N-methyl D-glycerol, and the like. Particularly preferred organiccations are 3-carbon, 4-carbon, 5-carbon and 6-carbon organic cations.Metallic cations useful in the practice of the present invention includebut are not limited to aluminum, calcium, lithium, magnesium, potassium,sodium, zinc, iron, ammonium, and the like. Particularly preferredcations include sodium, potassium, choline, lithium, meglumine,D-lysine, ammonium, magnesium, and calcium.

Specific examples of osmotically active salts that may be used with thesodium channel blockers described herein to carry out the presentinvention include, but are not limited to, sodium chloride, potassiumchloride, choline chloride, choline iodide, lithium chloride, megluminechloride, L-lysine chloride, D-lysine chloride, ammonium chloride,potassium sulfate, potassium nitrate, potassium gluconate, potassiumiodide, ferric chloride, ferrous chloride, potassium bromide, etc.Either a single salt or a combination of different osmotically activesalts may be used to carry out the present invention. Combinations ofdifferent salts are preferred. When different salts are used, one of theanion or cation may be the same among the differing salts.

Osmotically active compounds of the present invention also includenon-ionic osmolytes such as sugars, sugar-alcohols, and organicosmolytes. Sugars and sugar-alcohols useful in the practice of thepresent invention include but are not limited to 3-carbon sugars (e.g.,glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both the D and Lforms of erythrose, threose, and erythrulose); 5-carbon sugars (e.g.,both the D and L forms of ribose, arabinose, xylose, lyxose, psicose,fructose, sorbose, and tagatose); and 6-carbon sugars (e.g., both the Dand L forms of altose, allose, glucose, mannose, gulose, idose,galactose, and talose, and the D and L forms of allo-heptulose,allo-hepulose, gluco-heptulose, manno-heptulose, gulo-heptulose,ido-heptulose, galacto-heptulose, talo-heptulose). Additional sugarsuseful in the practice of the present invention include raffinose,raffinose series oligosaccharides, and stachyose. Both the D and L formsof the reduced form of each sugar/sugar alcohol useful in the presentinvention are also active compounds within the scope of the invention.For example, glucose, when reduced, becomes sorbitol; within the scopeof the invention, sorbitol and other reduced forms of sugar/sugaralcohols (e.g., mannitol, dulcitol, arabitol) are accordingly activecompounds of the present invention.

Osmotically active compounds of the present invention additionallyinclude the family of non-ionic osmolytes termed “organic osmolytes.”The term “organic osmolytes” is generally used to refer to moleculesused to control intracellular osmolality in the kidney. See e.g., J. S.Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg,Am. J. Physiol. 268, F983-F996 (1995), each incorporated herein byreference. Although the inventor does not wish to be bound to anyparticular theory of the invention, it appears that these organicosmolytes are useful in controlling extracellular volume on theairway/pulmonary surface. Organic osmolytes useful as active compoundsin the present invention include but are not limited to three majorclasses of compounds: polyols (polyhydric alcohols), methylamines, andamino acids. The polyol organic osmolytes considered useful in thepractice of this invention include, but are not limited to, inositol,myo-inositol, and sorbitol. The methylamine organic osmolytes useful inthe practice of the invention include, but are not limited to, choline,betaine, carnitine (L-, D- and DL forms), phosphorylcholine,lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatinephosphate. The amino acid organic osmolytes of the invention include,but are not limited to, the D- and L-forms of glycine, alanine,glutamine, glutamate, aspartate, proline and taurine. Additionalosmolytes useful in the practice of the invention include tihulose andsarcosine. Mammalian organic osmolytes are preferred, with human organicosmolytes being most preferred. However, certain organic osmolytes areof bacterial, yeast, and marine animal origin, and these compounds arealso useful active compounds within the scope of the present invention.

Under certain circumstances, an osmolyte precursor may be administeredto the subject; accordingly, these compounds are also useful in thepractice of the invention. The term “osmolyte precursor” as used hereinrefers to a compound which is converted into an osmolyte by a metabolicstep, either catabolic or anabolic. The osmolyte precursors of thisinvention include, but are not limited to, glucose, glucose polymers,glycerol, choline, phosphatidylcholine, lyso-phosphatidylcholine andinorganic phosphates, which are precursors of polyols and methylamines.Precursors of amino acid osmolytes within the scope of this inventioninclude proteins, peptides, and polyamino acids, which are hydrolyzed toyield osmolyte amino acids, and metabolic precursors which can beconverted into osmolyte amino acids by a metabolic step such astransamination. For example, a precursor of the amino acid glutamine ispoly-L-glutamine, and a precursor of glutamate is poly-L-glutamic acid.

In one embodiment of this invention, mucolytic agents are utilized toprovide access to other therapeutic agents through the mucus layer tothe airway epithelium. Mucus forms a diffusion barrier which can preventtherapeutic molecules from reaching their intended site of action.

The access of the following therapeutic agents to their site of actionin the airway epithelium could be enhanced by the pre- or co-treatmentwith the mucolytic agents described in this invention.

Sodium Channel Blockers:

Coordinated ion transport by the airway epithelia directly regulates thehydration level of the mucosal surface. Importantly, sodium absorptionthrough the epithelial sodium channel (ENaC) provides the rate-limitingstep in hydration. In human subjects with loss of function mutation inENaC have ‘wet’ airway surfaces and extraordinarily fast mucousclearance (Kerem et al., N Engl J Med. 1999 July 15; 341(3):156-62).Conversely, increased sodium absorption through ENaC has been shown tobe the underlying cause of mucous dehydration and the formation ofmucous plugs in the lungs CF patients. Furthermore, transgenic mice thatoverexpress ENaC in the lungs have dehydrated airway surfaces andreduced/absent mucous clearance that results in death (Hummler et al.,Proc Natl Acad Sci USA. 1997 Oct. 14; 94(21):11710-5). As predicted fromclinical and experimental data, pharmacological blockade of ENaCconserves liquid on airway surfaces and increases mucus clearance (Hirshet al., J Pharmacol Exp Ther. 2008; 325(1):77-88). Particular examplesinclude, but are not limited to:

Small Molecule Channel Blockers:

Small molecule ENaC blockers are capable of directly preventing sodiumtransport through the ENaC channel pore. ENaC blocker that can beadministered by the methods of this invention include, but are notlimited to, amiloride, benzamil, phenamil, and amiloride analogues asexemplified by U.S. Pat. Nos. 6,858,614, 6,858,615, 6,903,105,6,995,160, 7,026,325, 7,030,117, 7,064,129, 7,186,833, 7,189,719,7,192,958, 7,192,959, 7,241,766, 7,247,636, 7,247,637, 7,317,013,7,332,496, 7,345,044, 7,368,447, 7,368,450, 7,368,451, 7,375,107,7,399,766, 7,410,968, 7,820,678, 7,842,697, 7,868,010, 7,875,619,7,956,059, 8,008,494, 8,022,210, 8,124,607, 8,143,256, 8,163,758,8,198,286, 8,211,895, 8,324,218, 8,507,497, 8,575,176, 8,669,262,7,956,059, 8,008,494, 8,022,210, 8,124,607, 8,143,256, 8,163,758,8,198,286, 8,211,895, 8,324,218 8,507,497 8,575,176, 8,669,262,7,956,059, 8,008,494, 8,022,210, U.S. Patent Application Publication No.US2014/0142118-A1, U. S. Patent Application No. US20140170244-A1, and U.S. Patent Application No. US20140171447-A1.

Protease Inhibitors:

ENaC proteolysis is well described to increase sodium transport throughENaC. Protease inhibitor block the activity of endogenous airwayproteases, thereby preventing ENaC cleavage and activation. Proteasethat cleave ENaC include furin, meprin, matriptase, trypsin, channelassociated proteases (CAPs), and neutrophil elastases. Proteaseinhibitors that can inhibit the proteolytic activity of these proteasesthat can be administered by the methods of this invention include, butare not limited to, camostat, prostasin, furin, aprotinin, leupeptin,and trypsin inhibitors.

Nucleic Acids and Small Interfering RNAs (siRNA):

Any suitable nucleic acid (or polynucleic acid) can be used to carry outthe present invention, including but not limited to antisenseoligonucleotide, siRNA, miRNA, miRNA mimic, antagomir, ribozyme,aptamer, and decoy oligonucleotide nucleic acids. See, e.g., US PatentApplication Publication No. 20100316628. In general, such nucleic acidsmay be from 17 or 19 nucleotides in length, up to 23, 25 or 27nucleotides in length, or more.

Any suitable siRNA active agent can be used to carry out the presentinvention. Examples include, but are not limited to, those described inU.S. Pat. No. 7,517,865 and US Patent Applications Nos. 20100215588;20100316628; 20110008366; and 20110104255. In general, the siRNAs arefrom 17 or 19 nucleotides in length, up to 23, 25 or 27 nucleotides inlength, or more.

Secretogogues:

Mutations in the cystic fibrosis (CF) gene result in abnormal iontransport across the respiratory epithelium (Matsui et al., Cell 1998;95:1005-15). Excessive absorption of sodium and the inability to secretechloride by the airway epithelium in patients with CF drives waterabsorption down an osmotic gradient generated by inappropriate saltabsorption, dehydrating airway mucous secretions and reducing the volumeof liquid in the PCL. In COPD, cigarette smoke impairs CFTR function,thus creating an acquired phenotype similar to CF.

P2Y₂ Receptor Agonists:

Agents that that may be administered in combination with the methods andmolecules described in the present invention include a group of P2Y₂agonists. Purinergic (P2Y₂) receptors are abundant on luminal surface ofhuman bronchial epithelium (HBE) and are known to stimulate CF secretionand inhibit Na⁺ absorption (Goralski et al., Curr Opin Pharmacol. 2010June; 10(3):294-9). UTP is an example of an endogenous P2Y₂ receptoragonist that provides a robust stimulation of chloride secretion,inhibition of sodium absorption and increase in airway surface liquidlayer in airway epithelium, thus increasing the mucus clearance which isthe primary defense mechanism of the lung. Early studies usinguridine-5-triphosphate (UTP) delivered via aerosol to airway surfaces ofCF and primary cilia dyskinesia (PCD) patients suggested the usefulnessof UTP in enhancing MC and improving mean cough clearance rates.

Suitable P2Y₂ receptor agonists are described in, but are not limitedto, U.S. Pat. Nos. 6,264,975, 5,656,256, 5,292,498, 6,348,589,6,818,629, 6,977,246, 7,223,744, 7,531,525 and U.S. Pat. AP.2009/0306009 each of which is incorporated herein by reference.

Activators of Alternative Chloride Channels such as CaCCs and ClC-2Class Channels:

CaCCs are broadly expressed in mammalian cells where they are involvedin a wide range of physiological functions, including transepithelialfluid secretion, oocyte fertilization, olfactory and sensory signaltransduction, smooth muscle contraction, and neuronal and cardiacexcitation. Whole cell current analysis indicates several commonfeatures between CaCC subfamilies, including slow activation followingmembrane depolarization, outwardly rectifying steady state currents andgreater iodide than chloride permeability. Single channel analysis hassuggested four or more distinct CaCC subclasses, with a wide range ofreported single channel conductances from less than 2 pS in cardiacmyocytes to 50 pS in airway epithelial cells.

The consequences of CaCC activation are cell type specific, for example,chloride secretion in epithelial cells, action potential generation inolfactory receptor neurons, smooth muscle contraction, and prevention ofpolyspermia in oocytes. In some cell types, such as smooth muscle cells,membrane depolarization activates voltagegated calcium channels,increasing intracellular calcium concentration. Although CaCCs werefunctionally characterized nearly three decades ago, their molecularidentity has remained unclear until recently, with potential candidatesincluding bestrophins (BEST1-BEST4) (Sun et al., Proc Natl Acad Sci USA99, 4008-4013 (2002) and Tsunenari et al., J Biol Chem 278, 41114-41125(2003)), the calcium activated chloride channel ClCA family proteins(Gruber et al., Genomics 1998; 54:200-214) and ClC3 (Huang P et al.(2001) Regulation of human CLC-3 channels by multifunctionalCa2+/calmodulin-dependent protein kinase. JBC 276: 20093-100). Threeindependent laboratories have identified TMEM16A, also calledanoctamin1, as a strong candidate for a CaCC (Yang Y D et al. (2008)TMEM16A confers receptor-activated calcium-dependent chlorideconductance. Nature. 455: 1210-15; Caputo A et al. (2008) TMEM16A, amembrane protein associated with calcium-dependent chloride channelactivity. Science. 322: 590-4; Schroeder B C et al. (2008) Expressioncloning of TMEM16A as a calcium-activated chloride channel subunit.Cell. 134: 1019-29). Three different strategies were used: databasesearching for membrane proteins with multiple transmembrane segments andunknown function (Yang Y D et al. (2008) TMEM16A confersreceptor-activated calcium-dependent chloride conductance. Nature. 455:1210-15), functional genomics following the observation that interleukin4 (I14) treated bronchial epithelial cells show increased CaCC activity(Caputo A et al. (2008) TMEM16A, a membrane protein associated withcalcium-dependent chloride channel activity. Science. 322: 590-4), andexpression cloning using axolotl oocytes that do not have endogenousCaCC activity (Schroeder B C et al. (2008) Expression cloning of TMEM16Aas a calcium-activated chloride channel subunit. Cell. 134: 1019-29).There is strong evidence to suggest TMEM16A is a key component of CaCC,including similarity to native CaCCs in its electrophysiologicalproperties, appearance of CaCC currents in various transfected cellsystems, reduction in CaCC currents following RNAi knockdown, and itstissue distribution. TMEM16A has eight putative transmembrane segmentswithout domains evidently involved in calcium regulation.

ClC2 is a ubiquitously expressed, inwardly rectifying chloride channelthat is activated by cell swelling. ClC2 was thought to be involved incell volume regulation, but it has different biophysical characteristicsfrom the volume sensitive chloride channels that have been characterizedin many tissues. Suitable alternative chloride channel activators aredescribed in U.S. Pat. Nos. 6,015,828, 6,159,969 and 7,253,295. Thetherapeutic efficacy of activators of Alternative Chloride Channels suchas CaCCs and ClC-2 Class Channels can be enhanced by the administrationof compounds and methods of this invention.

Modulators of CFTR Activity

The hereditary lethal disease cystic fibrosis is caused mutations in thegene encoding CFTR protein, a cAMP activated chloride channel expressedin the airway epithelia. Various mutations in CFTR cause ion transportdysfunction by limiting the chloride ion secretion to the surface of theairway epithelium via CFTR and by dys-regulation of sodium ionabsorption, leading to excessive absorption of sodium cations. Thesedefects in ion transport result in impaired hydration of airway surfaceliquid layer, decrease in mucus clearance and lead to progressive lossof lung function. Recently, it has been shown that CFTR functionaldefects are present in cigarette smoke exposed tissue, thus implying therole of CFTR dysfunction in COPD.

Over 1500 putative mutations have been described in CFTR, which can bedivided into classes according to the molecular mechanism of the geneticdefect (Rowe et al., Pulm Pharmacol Ther., 23(4):268-78 (2010)). Anunderstanding of the biology of each of these mutations has led totherapeutic strategies based on the particular mutation type. Class Imutations include premature termination codons (PTCs, e.g. nonsensemutations) within the coding region of CFTR, which cause prematuretruncation of normal protein translation. These mutations are found in10% of CF patients, but are particularly common in Ashkenazi Jews (75%of mutant CFTR alleles). Class II CFTR mutations include F508del CFTR,the most common mutation in humans (accounting for 75% of alleles andfound in approximately 90% of CF patients). The deletion ofphenylalanine at the 508 position causes CFTR to exhibit abnormalfolding characterized by deficient stabilization by domain-domaininteractions between the nucleotide binding domain 1 (NBD1) and thetransmembrane domains. The misfolded protein is recognized by cellularchaperones within the endoplasmic reticulum (ER), directed to theproteasome, and rapidly degraded prior to reaching its active site atthe cell surface. Because the cellular machinery responsible for therecognition and degradation of the misfolded protein is not 100%efficient, particular individuals exhibit low levels of surfaceexpression of F508del CFTR, which may account for partial CFTR activity(and a more mild CF phenotype) observed in individuals homozygous forF508del CFTR, and could represent a population more amenable to proteinrepair. Even when at the cell surface, F508del CFTR exhibits reducedgating, suggesting that misfolded CFTR also exhibits reduced CFTR ionchannel activity. Class III and IV CFTR mutations are characterized byfull-length CFTR that reaches the cell surface but exhibit reduced iontransport activity owing to abnormal channel gating (Class III, e.g.G551D) or reduced conductivity of the ion channel pore (Class IV, e.g.R117H). Similarly, splicing mutants (Class V) and mutations within theC-terminus (Class VI) are also full length, but exhibit reduced activityowing to reduced numbers of active channels within the plasma membrane.Although the molecular basis of CFTR mutants is complex and as yetincomplete, the classification of CFTR mutants can be simplified intothe therapeutically relevant groups based on the activity of agents indevelopment. Both traditional and high-throughput drug discoveryprograms have resulted in discovery of novel compounds that addressspecific mutant CFTR alleles. These ‘CFTR modulators’ arepharmacological agents intended to repair the CFTR protein and aredescribed in each section that follows.

Potentiators of cell-surface cystic fibrosis transmembrane conductanceregulator CFTR mutation classes that result in dysfunctional CFTR thatresides at the plasma membrane include Class III, IV, V, and VImutations and represent potential targets for CFTR activators. G551DCFTR represents an archetype CFTR allele for this category of agents, asit exhibits normal surface expression and half-life, but confers asevere defect in channel gating owing to an amino acid substitution inthe adenosine triphosphate (ATP) binding pocket within the nucleotidebinding domains (Gregory, R. J. et al. (1991) Maturation and function ofcystic fibrosis transmembrane conductance regulator variants bearingmutations in putative nucleotide-binding domains 1 and 2. MCB 11:3886-93; Bompadre, S. G. et al. (2007) G551D and G1349D, twoCF-associated mutations in the signature sequences of CFTR, exhibitdistinct gating defects. Gen Physiol. 129: 285-298). Flavonoids are wellknown activators of mutant CFTR and were among the first to be studiedfor beneficial effects in human individuals (including topicaladministration). Although agents such as genistein were affected by lackof efficacy in the nasal airway, more recent efforts have demonstratedactivity of the flavonoid quercetin in the nose. However, flavonoidagents are challenged by poor solubility and systemic absorption, andare poor development candidates for inhaled therapeutics. More recentdiscovery strategies have focused on identification of compounds that‘potentiate’ CFTR activity, restoring endogenous regulation (e.g. cyclicadenosine monosphosphate (cAMP)-dependent regulation) and ion transportwithout excessive, constitutive activation that may potentially bedetrimental (such as excessive CFTR activation seen with certaindiarrheal illnesses). Identification of agents of this type is amenableto high-throughput screening-based strategies to discover agents thatactivate mutant CFTR by measuring the effects on anion conductance incell-based screening assays. A number of specific strategies have beenused for screens of this sort, including chloride sensitive dyes,fluorescence resonance energy transfer-based analysis of membranepotential, and cell conductance of airway monolayers. Identification andcharacterization of small molecule potentiators of mutant CFTR have ledto the development of agents with pronounced activity in vitro and inthe clinic.

Significant effort has been directed toward the goal of correcting thefolding of F508del CFTR, thus restoring ion channel activity to themisfolded protein. A diverse array of cellular targets have beenexplored, commensurate with the large number of proteins now known tointeract with CFTR biogenesis. Agents such as 4-phenyl butyratedownregulate Hsc70 (or other cell chaperones) central to the foldingprocess, and represent an early example of compounds tested in theclinic. Other more recent efforts have resulted from high-throughputlibrary screens for chloride channel function following incubation oftest compounds with F508del expressing cells. A number of thesestrategies have identified F508del correctors that may address cellbiogenesis through chaperone pathways. Pharmacologic activity of suchagents has also been reported to augment F508del CFTR half-life in theplasma membrane through altered surface recycling attributed to featuresof the cellular processing machinery or reduced endocytic trafficking.This class of agents may be potential drug development candidates iftheir safety in vivo is confirmed. Other compounds have been shown todirectly interact with CFTR and may offer greater specificity thanagents that alter general aspects of cell folding or cellular qualitycontrol. The global cellular response to misfolded protein may alsorepresent a target. Histone deacetylases (HDAC) have far-ranging effectson gene expression, and specific members of the HDAC family are involvedin the ER associated degradation pathway promoting degradation ofF508del CFTR. Treatment of CF cells with HDAC inhibitors can modulate ERstress, and HDACs such as suberoylanilidehydroxamic acid, as well assiRNA-silencing of HDACs, increase levels of F508del CFTR in the cellmembrane. The combination of approaches such as these reveal a number ofpotential pharmacologic agents for F508del correction. Additive orsynergistic rescue of F508del CFTR using more than one such strategy mayoffer hope of achieving ion transport activity sufficient to confer anormal phenotype in CF respiratory epithelia.

Read-through of premature termination codons (PTCs) represents anotherexciting approach to address the underlying cause of CF, and many othergenetic diseases caused by PTCs. Certain aminoglycosides and otheragents have the capacity to interact with the eukaryotic rRNA within theribosomal subunits. Although this interaction is much weaker than thatseen in prokaryotes and is distinct from the primary cause ofaminoglycoside toxicity in human individuals, it can modestly reduce thefidelity of eukaryotic translation by interrupting the normalproofreading function of the ribosome. Insertion of a near cognate aminoacid at a premature stop codon allows protein translation to continueuntil one of several stop codons normally present at the end of the mRNAtranscript is reached and properly utilized. The specificity of thestrategy has been attributed to greater stop codon fidelity at theauthentic end of mRNA and has been established in vitro by demonstratingno detectable elongation beyond native stop codons.

CFTR activity modulating compounds that can be administered incombination with the methods and molecules described in the presentinvention include, but are not limited to, compounds described in US2009/0246137 A1, US 2009/0253736 A1, US 2010/0227888 A1, U.S. Pat. No.7,645,789, US 2009/0246820 A1, US 2009/0221597 A1, US 2010/0184739 A1,US 2010/0130547 A1, US 2010/0168094 A1, U.S. Pat. Nos. 7,553,855,7,772,259 B2, U.S. Pat. No. 7,405,233 B2, US 2009/0203752, and U.S. Pat.No. 7,499,570.

Anti-Infective Agents:

Chronic obstructive pulmonary diseases are accompanied by both acute andchronic bacterial infections. Both acute and chronic infections lead tochronic inflammation that has acute flare-ups in the form of pulmonaryexacerbations. The underlying inflammation is treated with variety ofinhaled anti-inflammatory agents. For example, in cystic fibrosis themost common bacteria causing chronic infection is Pseudomonas aeruginosa(P. aeruginosa) and antibiotics that are effective against this bacteriaare a major component of treatment (Flume, Am J Respir Crit Care Med.176(10):957-69 (2007)). Also bacteria such as Staphylococcus aureus (S.aureus), Burkholderia cepacia (B. cepacia) and other gram negativeorganisms as well as anaerobes are isolated from respiratory secretionsand people with CF may benefit from treatment of these pathogens tomaintain their lung health. Anaerobic bacteria are also recognized as afeature of CF airways, sinuses in subjects with chronic sinusitis, andlikely airways of subjects with COPD. Similarly, aspirations ormicroaspirations, especially in elderly population and during sleep, areassociated with a chemical pneumonitis, anaerobic infections andsubsequent bronchiectasis. An ideal treatment of aspiration-relatedpneumonitis and anaerobic infection would be an immediate treatment. Assuch, antibiotics are used to eradicate early infections, duringpulmonary exacerbations and as chronic suppressive therapy.

The primary measure of antibiotic activity is the minimum inhibitoryconcentration (MIC). The MIC is the lowest concentration of anantibiotic that completely inhibits the growth of a microorganism invitro. While the MIC is a good indicator of the potency of anantibiotic, it indicates nothing about the time course of antimicrobialactivity. PK parameters quantify the lung tissue level time course of anantibiotic. The three pharmacokinetic parameters that are most importantfor evaluating antibiotic efficacy are the peak tissue level (Cmax), thetrough level (Cmin), and the Area Under the tissue concentration timeCurve (AUC). While these parameters quantify the tissue level timecourse, they do not describe the killing activity of an antibiotic.

Integrating the PK parameters with the MIC gives us three PK/PDparameters which quantify the activity of an antibiotic: the Peak/MICratio, the T>MIC, and the 24 h-AUC/MIC ratio. The Peak/MIC ratio issimply the Cpmax divided by the MIC. The T>MIC (time above MIC) is thepercentage of a dosage interval in which the serum level exceeds theMIC. The 24 h-AUC/MIC ratio is determined by dividing the 24-hour-AUC bythe MIC. The three pharmacodynamic properties of antibiotics that bestdescribe killing activity are time-dependence, concentration-dependence,and persistent effects. The rate of killing is determined by either thelength of time necessary to kill (time-dependent), or the effect ofincreasing concentrations (concentration-dependent). Persistent effectsinclude the Post-Antibiotic Effect (PAE). PAE is the persistentsuppression of bacterial growth following antibiotic exposure.Using these parameters, antibiotics can be divided into 3 categories:

Goal of PK/PD Pattern of Activity Antibiotics Therapy Parameter Type IAminoglycosides Maximize 24 h- Concentration- Daptomycin concentrationsAUC/MIC dependent killing and Fluoroquinolones Peak/MIC Prolongedpersistent Ketolides effects Type II Carbapenems Maximize T > MICTime-dependent killing Cephalosporins duration of and Erythromycinexposure Minimal persistent Linezolid effects Penicillins Type IIIAzithromycin Maximize 24 h- Time-dependent killing Clindamycin amount ofAUC/MIC and Oxazolidinones drug Moderate to prolonged Tetracyclinespersistent effects. Vancomycin

For Type I antibiotics (AG's, fluoroquinolones, daptomycin and theketolides), the ideal dosing regimen would maximize concentration,because the higher the concentration, the more extensive and the fasteris the degree of killing. Therefore, the 24 h-AUC/MIC ratio, and thePeak/MIC ratio are important predictors of antibiotic efficacy. Foraminoglycosides, it is best to have a Peak/MIC ratio of at least 8-10 toprevent resistance. For fluoroquinolonesys gram negative bacteria, theoptimal 24 h-AUC/MIC ratio is approximately 125. Versus gram positives,40 appears to be optimal. However, the ideal 24 h-AUC/MIC ratio for FQ'svaries widely in the literature.

Type II antibiotics (beta-lactams, clindamycin, erythromcyin,carbapenems and linezolid) demonstrate the complete opposite properties.The ideal dosing regimen for these antibiotics maximizes the duration ofexposure. The T>MIC is the parameter that best correlates with efficacy.For beta-lactams and erythromycin, maximum killing is seen when the timeabove MIC is at least 70% of the dosing interval.

Type III antibiotics (vancomycin, tetracyclines, azithromycin, and thedalfopristin-quinupristin combination) have mixed properties, they havetime-dependent killing and moderate persistent effects. The ideal dosingregimen for these antibiotics maximizes the amount of drug received.Therefore, the 24 h-AUC/MIC ratio is the parameter that correlates withefficacy. For vancomycin, a 24 h-AUC/MIC ratio of at least 125 isnecessary.

Patients including, but not limited to, CF, COPD, non-CF bronchiectasis,aspiration pneumonia, asthma and VAP patients suffering from respiratoryinfection caused by bacteria susceptible to meropenem may benefit fromsuch treatment. Examples of carbapenam antibiotics are: imipenam,panipenam, meropenam, doripenem, biapenam, MK-826, DA-1131, ER-35786,lenapenam, S-4661, CS-834 (prodrug of R-95867), KR-21056 (prodrug ofKR-21012), L-084 (prodrug of LJC 11036) and CXA-101. The therapeuticefficacy of all antiinfective agents described can be enhanced by thepre- or co-administration of compounds and methods of this invention.

Exemplary Anti-Inflammatory Agents:

Inhaled corticosteroids are the standard of chronic care for asthma,COPD and other respiratory diseases characterized by acute and chronicinflammation leading to airflow limitation. Examples ofanti-inflammatory agents suitable for administration in combination withthe methods and molecules described in the present invention includebeclomethasone, budesonide, and fluticasone and a group ofanti-inflammatory medications that do not contain steroids known asnon-steroiodal anti-inflammatory drugs (NSAIDs).

Products of arachidonic acid metabolism, specifically the leukotrienes(LTs), contribute to pulmonary inflammation. Cysteinylleukotrienes(LTC4, LTD4, and LTE4) are produced predominantly by eosinophils, mastcells, and macrophages. Examples of leukotriene modifiers suitable foradministration by the method of this invention include monteleukast,zileuton and zafirlukast.

Mast cell stabilizers are cromone medications such as cromolyn (sodiumcromoglycate) used to prevent or control certain allergic disorders.They block a calcium channel essential for mast cell degranulation,stabilizing the cell and thereby preventing the release of histamine andrelated mediators. As inhalers they are used to treat asthma, as nasalsprays to treat hay fever (allergic rhinitis) and as eye drops forallergic conjunctivitis. Finally, in oral form they are used to treatthe rare condition of mastocytosis.

PDE4 inhibitors have been shown to modulate pulmonary inflammation andused for treatment of chronic obstructive pulmonary diseases. Examplesof PDE4 inhibitors suitable for use in combination with the methods andmolecules described in the present invention include, but is not limitedto theophylline and roflumilast.

Exemplary Bronchodilators:

Nitric Oxide (NO) Donors:

NO, NO Donors, NO and Peroxynitrite Scavengers and Inducible NO SynthaseActivity Modulators. Nitric oxide is a potent endogenous vasodilator andbronchodilator that can be exogenously administered via inhalation. Itis synthesized by the conversion of the terminal guanidine nitrogen atomof L-arginine via endothelial cell calcium dependent enzyme nitric oxidesynthetase and then diffuses across the cell membrane to activate theenzyme guanylatecyclase. This enzyme enhances the synthesis of cyclicguanosine monophosphate (cGMP), causing relaxation of vascular andbronchial smooth muscle and vasodilatation of blood vessels (Palmer,Circ Res., 82(8):852-61 (1998)).

Nitric oxide synthesised in endothelial cells that line blood vesselshas a wide range of functions that are vital for maintaining a healthyrespiratory and cardiovascular systems (Megson I L et al Expert OpinInvestig Drugs. 2002 May; 11(5):587-601.). Reduced nitric oxideavailability is implicated in the initiation and progression of manydiseases and delivery of supplementary nitric oxide to help preventdisease progression is an attractive therapeutic option. Nitric oxidedonor drugs represent a useful means of systemic nitric oxide deliveryand organic nitrates have been used for many years as effectivetherapies for symptomatic relief from angina. However, nitrates havelimitations and a number of alternative nitric oxide donor classes haveemerged since the discovery that nitric oxide is a crucial biologicalmediator.

In the respiratory tract, NO is produced by residential and inflammatorycells (Ricciardolo F L et al. Curr Drug Targets 2006 June; 7(6):721-35).NO is generated via oxidation of L-arginine that is catalysed by theenzyme NO synthase (NOS). NOS exists in three distinct isoforms:neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).NO derived from the constitutive isoforms of NOS (nNOS and eNOS) andother NO-adduct molecules (nitrosothiols) are able to modulatebronchomotor tone. NO derived from the inducible isoform of NO synthase,up-regulated by different cytokines via NF-kappaB-dependent pathway,seems to be a pro-inflammatory mediator with immunomodulatory effects.In aging CF patients, expression of iNOS is significantly reduced (Yoonet al., J Clin Invest. 2006 February; 116(2):436-46). This reducedexpression of iNOS in chronic CF is associated with emergence of mucoidmuc mutant subpopulation of P. aeruginosa. It has been suggested that 15mM NO₂ ⁻ kills mucA P. Aeruginosa in CF airways at pH 6.5. NO itself oras a precursor to iron-nitrosyl species has been implicated in thisantimicrobial effect. Therefore inhaled NO₂ ⁻, including but not limitedinhaled NaNO₂, has an appeal as a CF therapy. The production of NO underoxidative stress conditions secondarily generates strong oxidizingagents (reactive nitrogen species) that may amplify the inflammatoryresponse in asthma and COPD. Moreover, NO can be exhaled and levels areabnormal in stable atopic asthma and during exacerbations in both asthmaand COPD. Exhaled NO might therefore be a non-invasive tool to monitorthe underlying inflammatory process. It is suggested that NOS regulationprovides a novel target in the prevention and treatment of chronicinflammatory diseases of the airways such as asthma and COPD.

Examples of NO, NO donors and NO synthase activity modulators suitablefor administration in combination with the methods and moleculesdescribed in the present invention include inhaled NO, agents disclosedin Vallance et al. Fundam Clin Pharmacol. 2003 February; 17(1):1-10,Al-Sa'doni H H et al. Mini Rev Med Chem. 2005 March; 5(3):247-54, MillerM R et al. Br J Pharmacol. 2007 June; 151(3):305-21. Epub 2007 Apr. 2and Katsumi H et al. Cardiovasc Hematol Agents Med Chem. 2007 July;5(3):204-8.

Under certain conditions, inducible NO synthase activity leads tooverproduction of NO which in turn increases inflammation and tissueinjury. Under these conditions, the following inducible NO synthaseinhibitors, NO scavengers and peroxynitrite scavengers administered incombination with the methods and molecules described in the presentinvention are suitable: Bonnefous et al. J. Med. Chem., 2009, 52 (9), pp3047-3062, Muscara et al AJP-GI June 1999 vol. 276 no. 6 G1313-G1316 orHansel et al. FASEB Journal. 2003; 17:1298-1300.

Beta 2-Adrenergic Receptor Agonists:

It has been established that administration of super-therapeuticconcentrations of receptor agonists leads to receptor desensitizationand loss of efficacy. For example, this phenomenon has been describedfor beta 2-adrenoceptor based bronchodilator agents (Duringer et al., BrJ Pharmacol., 158(1):169-79 (2009)). High concentration of thesereceptor agonist agents leads to the receptor phosphorylation,internalization and potential degradation. Administration of receptoragonists, which cause tachyphylaxis following bolus administration viafast nebulizer, by inhalation over the course of 8 to 24 hours orovernight to a patient via nasal cannula improves the efficacy of suchagents due to decreased extent of tachyphylaxis. Beta 2-adrenergicreceptor agonsists include albuterol, levalbuterol, salbutamol,procaterol, terbutaline, pirbuterol, and metaproterenol. The therapeuticefficacy of beta 2-adrenergic receptor agonists can be enhanced by thepre- or co-administration of compounds and methods of this invention.

Exemplary Gene Carriers:

Examples of gene carriers for the administration of gene therapy includeviruses, DNA:protein complexes, plasmids, DNAs, and RNAs.

Other Exemplary Therapeutic Agents:

Examples of other classes of therapeutic agents suitable foradministration in combination with the methods and molecules describedin the present invention include antivirals such as ribavirin,anti-fungal agents such as amphotericin, intraconazol and voriconazol,immunosuppressants, anti-rejection drugs such as cyclosporine,tacrolimus and sirolimus, bronchodilators including but not limited toanticholinergic agents such as ipratropium, tiotropium, aclidinium andothers, PDE5 inhibitors siRNAs, gene therapy vectors, aptamers,endothelin-receptor antagonists, alpha-1-antitrypsin, prostacyclins,vaccines, PDE-4 and PDE-5 inhibitors and steroids such asbeclamethasone, budesonide, ciclesonide, flunisolide, fluticasone,memetasone and triamcinolone.

EXPERIMENTAL PROCEDURES AND BIOLOGICAL ASSAYS

Compounds of Formula I: Compounds of formula I are readily prepared bymethods well known in the art as exemplified and detailed below.

Mucin Agarose Gel Western Blots: Reducing agent stock solutions are madeup in 100 mM Potassium Phosphate and are buffered to pH 6.5. Thereducing agent stocks are diluted into saliva samples (pH 6.5) toachieve the final desired reducing agent concentration. Reactions areincubated at 25.0 for the desired time (0-120 minutes). The reactionsare quenched using at least a 2-fold excess of N-ethylmaleimide and/orhydrogen peroxide. A 5× concentrated sample loading buffer is dilutedinto the samples to achieve a 1× concentration (1×TAE, 5% glycerol, 0.1%SDS, Bromophenol Blue). Samples (50 ug) are analyzed by electrophoresison 0.9% agarose gels using a buffer system consisting of 1×TAE/0.1% SDS.The agarose gel is soaked for 15 min in 4×SSC (0.6 M NaCl, 60 mMTri-sodium Citrate dihydrate) containing 10 mM DTT before transferringthe samples from the gel onto a nitrocellulose membrane by vacuumblotter. Unreduced and reduced Muc5B are visualized using a polyclonalantibody directed towards Muc5B and a Protoblot II AP System withstabilized substrate. FIGS. 2-7 clearly show superiority of the dithiolcompounds of the present invention over DTT and NAC.

BiP Induction: Reducing agents are made up in Hanks Balanced SaltSolution (HBSS)/25 mM HEPES pH 7.4. Each compound solution (10 uL) isadded apically to primary hBEs for 6 hrs. The hBEs are lysed in RIPAbuffer supplemented with protease inhibitor cocktail (Roche) and 1 mMPMSF. The samples are normalized to contain the same total amount ofprotein followed by addition of 2×SDS sample buffer (100 mM Tris-HCl(pH6.8)/4% SDS/0.05% Bromophenol Blue/20% glycerol). Samples (20□g) areanalyzed by electrophoresis on a 10% SDS-PAGE gel and transferred to anitrocellulose membrane. BiP levels are visualized using a polyclonalantibody directed towards BiP and the LiCor Odyssey imaging detectionsystem. Thapsigargin (TG, 2.5 □M) served as a positive control for BiPinduction.

DTNB ASSAY: This assay determines the rate which a mucolytic agent canreduce a disulfide bond using 5,5-Dithiobis(2-nitrobenzoic acid). DTNBin various pH buffers and allows comparison of the kinetic rates ofreducing agents. First, reducing agent stock solutions (30 mM) were madeup in DMSO. Each compound stock solution was diluted, 1.5 ml in 1 ml 50mM Tris-HCl buffer, pH 7.5, and then added in a 1:1 volumetric ratio toa solution of 100 mM DTNB in 50 mM Tris-HCl buffer, pH 7.5. Max Abs₄₁₂was measured and then utilized to calculate the activity concentration.If the observed activity concentrations differed from expected activityconcentrations by more than 5%, the volume was accordingly adjusted tokinetically test the rate in the next step. After having added thereducing agents, diluted in a range of pH buffer solutions (pH 6.0-7.5),to 45 mM DTNB in 50 mM Tris-HCl and measured the Abs₄₁₂ for 5 min rateswere calculated as a 2^(nd) order kinetic. Table I summarizes theresults vs. NAC and DTT.

TABLE 1 Kinetic Reaction Rates of Reduction of DTNB by the dithiolCompounds of the present invention compared to DTT and NAC. pH Compound6 6.5 7 7.5 NAC 10 26 69 230 DTT 69 176 382 2088 I 285 745 1916 7265 J86 337 1043 2016 K 678 1820 3775 9143 L 1911 5565 8847 14566 C 2460 712212313 17317 N 952 2366 4382 9532 O 921 2500 5412 13547 P 1018 2701 618013243 Q 954 2984 6077 15927 R 2901 7382 13522 16870 S 4483 10715 1373215469 E 5933 13718 16393 17109 W 4850 — — — H 6835 — — — B 1491 — — — T5144 — — — U 5761 — — — X 8029 — — — Y 8718 — — — G 5154 — — — V 14194568 6192 10043 Z 7200 10420 10631 18513 KK 873 2411 5163 13896 LL 7432352 4661 8241

TMV Studies in Sheep

Test articles were administered to the pulmonary surfaces as aninhalation aerosol. For human neutrophil Elastase (HNE) studies,tracheal mucus velocity (TMV) was measured at 1 and 2 hours after HNE(but before drug); and 1, 30, 60, 90, and 120 minutes post-aerosoladministration of Compound G.

The measurements of TMV were made as follows: Eight to 10 radiopaqueTeflon disks (˜1 mm in diameter, 0.8 mm thick, and weighing between1.5-2 mg) were introduced into the trachea via an endotracheal tube(ETT). The particles were insufflated by a catheter connected to asource of compressed air (flow rate of 3-4 ml/min at 50 psi). Thecatheter was removed following insufflation without contacting thetracheal surface. To minimize the ETT effects on TMV, the cuff wasdeflated throughout the study except for the period of drug delivery.The disk movements were recorded using videotaped fluoroscopy, andindividual disk velocities were calculated by measuring the distancetraveled by each disk over a 60-s period. A collar containing radiopaquemarkers of predetermined length was placed around the animal's neck,which was used as a standard to correct for magnification effectsintrinsic to the fluoroscopy unit. The mean value of disk velocities wascalculated for each time point. To avoid dehydration, the sheep wereperiodically gavaged with tap water via a nastrogastric tube. To avoiddesiccation of the tracheal mucosa caused by sustained intubation, theinspired air was warmed and humidified by a Bennet Humidifier(Puritan-Bennett, Lenexa, Kans.).

The activity of Compound G (1 μmole/kg) was subsequently tested in thissheep model of “acute bronchitis” in which neutrophil elastase (NE) wasadministered via aerosol prior to dosing with Compound G. In sheeptreated with NE, Compound G, but not vehicle, restored TMV to normal,pre-NE levels, which was sustained for 2 h post-dosing.

1. Preparation of S,S′-(4-(2-aminoethoxy)-1,2-phenylene)bis(methylene)diethanethioate hydrochloride(A)

Preparation of Dimethyl 4-hydroxyphthalate (2)

A solution of 4-hydroxyphthalic acid 1 (25.0 g, 137 mmol) in MeOH (700mL) was charged with SOCl₂ (30.0 mL, 412 mmol) at 0° C. and stirred atroom temperature for 20 h. The solvent was removed and the residue waspartitioned between saturated aqueous NaHCO₃ solution (100 mL) andCH₂Cl₂ (250 mL). The CH₂Cl₂ layer was separated and the aqueous layerwas extracted with CH₂Cl₂ (2×250 mL). The combined organic extracts werewashed with brine, dried over Na₂SO₄, and concentrated to affordcompound 2 (26.5 g, 92%) as a colorless oil: ¹H NMR (400 MHz, CDCl₃) δ7.72 (d, J=8.5 Hz, 1H), 7.00 (d, J=2.6 Hz, 1H), 6.91 (dd, J=8.5, 2.6 Hz,1H), 3.89 (s, 3H), 3.85 (s, 3H).

Preparation of Dimethyl4-{2-[(tert-butoxycarbonyl)amino]ethoxy}phthalate (4)

A solution of compound 2 (6.00 g, 28.6 mmol) in DMF (30 mL) was chargedwith K₂CO₃ (15.7 g, 114 mmol) and stirred at room temperature for 5 min.The above reaction mixture was charged with compound 3 (9.98 g, 42.9mmol) and the final reaction mixture was stirred at room temperature for72 h. Water (300 mL) was added to the reaction mixture and extractedwith CH₂Cl₂ (2×300 mL). The combined organic extracts were concentratedand the residue was purified by column chromatography (silica gel, 20%to 30% EtOAc in hexanes) to afford compound 4 (9.00 g, 89%) as a yellowoil: ¹H NMR (400 MHz, DMSO-d₆) δ 7.78 (d, J=8.4 Hz, 1H), 7.17 (d, J=2.5Hz, 1H), 7.01 (dd, J=8.4, 2.5 Hz, 1H), 7.01 (t, J=6.0 Hz, 1H), 4.08 (t,J=5.5 Hz, 2H), 3.80 (s, 3H), 3.78 (s, 3H), 3.31 (t, J=6.4 Hz, 2H), 1.37(s, 9H).

Preparation of Tert-butyl{2-[3,4-bis(hydroxymethyl)phenoxy]ethyl}carbamate (5A solution ofcompound 4 (9.00 g, 25.5 mmol) in THF (200 mL) was charged with lithiumaluminum hydride (1 M solution in diethyl ether, 102 mL, 102 mmol) at 0°C. The resulting reaction mixture was stirred at 0° C. for 1 h andquenched with ice-cold water at 0° C. The reaction mixture was dilutedwith chloroform (300 mL) and filtered through a Celite pad, and theCelite pad was washed with chloroform (2×300 ml). The filtrate wasconcentrated under vacuum to afford 5 (6.80 g, 90%) as a yellow oil: ¹HNMR (400 MHz, CDCl₃) δ 7.23 (d, J=8.3 Hz, 1H), 6.89 (d, J=2.7 Hz, 1H),6.78 (dd, J=8.3, 2.7 Hz, 1H), 5.10-5.01 (m, 1H), 4.65 (s, 2H), 4.64 (s,2H), 3.99 (t, J=5.3 Hz, 2H), 3.49 (dd, J=10.6, 5.3 Hz, 2H), 1.44 (s,9H).

Preparation of(4-{2-[(tert-butoxycarbonyl)amino]ethoxy}-1,2-phenylene)bis(methylene)dimethanesulfonate(6)

A solution of 5 (20.0 g, 67.3 mmol) in CH₂Cl₂ (500 mL) was charged withEt₃N (73.5 mL, 538 mmol) followed by methanesulfonyl chloride (20.8 mL,270 mmol) at 0° C. and stirred at room temperature for 22 h. Water (200mL) was added to the reaction mixture and extracted with CH₂Cl₂ (3×200mL) The combined organic extracts were washed with brine, dried overNa₂SO₄, and concentrated to afford compound 6 (27.0 g, crude) as a brownoil, which was directly used for the next step without furtherpurification.

Preparation of Compound 7

A solution of compound 6 (27.0 g, crude, 67.3 mmol) in a mixture of THF(200 ml) and DMF (40 mL) was charged with KSAc (19.2 g, 168 mmol) andstirred at room temperature for 20 h. The solvent was removed underreduced pressure and the reaction mixture was partitioned between water(100 mL) and CH₂Cl₂ (250 mL). The CH₂Cl₂ layer was separated and theaqueous layer was extracted with CH₂Cl₂ (2×300 mL). The combined organicextracts were concentrated and the residue was purified by columnchromatography (silica gel, 10% to 20% EtOAc in hexanes) to affordcompound 7 (16.2 g, 58% over two steps) as a yellow solid: ¹H NMR (400MHz, CDCl₃) δ 7.21 (d, J=8.5 Hz, 1H), 6.84 (d, J=2.6 Hz, 1H), 6.72 (dd,J=8.5, 2.6 Hz, 1H), 5.05-4.93 (m, 1H), 4.11 (s, 4H), 3.97 (t, J=5.2 Hz,2H), 3.50 (dd, J=10.8, 6.1 Hz, 2H), 2.35 (s, 3H), 2.33 (s, 3H), 1.44 (s,9H).

Preparation of compound A;S,S′-(4-(2-aminoethoxy)-1,2-phenylene)bis(methylene) diethanethioatehydrochloride

Compound 7 (6.00 g, 14.5 mmol) was dissolved in 4 N HCl in dioxane (50mL) at room temperature, and the solution was stirred at sametemperature for 2 h. After removal of the solvent, the residue wastriturated with MTBE to afford hydrochloric acid salt A (4.50 g, 88%) asan off-white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.24 (d, J=8.6 Hz, 1H),6.97 (d, J=2.8 Hz, 1H), 6.85 (dd, J=8.6, 2.8 Hz, 1H), 4.20 (dd, J=4.9,4.3 Hz, 2H), 4.14 (s, 2H), 4.13 (s, 2H), 2.35 (t, J=4.9 Hz, 2H), 2.32(s, 3H), 2.31 (s, 3H).

2. Preparation of(R)-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)-6-guanidinohexanamideHydochloride

Preparation of Compound 9

Compound A (450 mg, 1.26 mmol) and acid 8 (566 mg, 1.26 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (0.88 mL, 5.04 mmol) andHATU (479 mg, 1.26 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. The solvent was removed under reducedpressure, the residue was dissolved in CH₂Cl₂ (100 mL), and the solutionwas quickly washed with saturated aqueous NaHCO₃ (2×50 mL) followed bybrine (50 mL).

The organic layer was dried over Na₂SO₄ and concentrated. The residuewas purified by column chromatography (silica gel, 50% to 80% EtOAc inhexanes) to afford compound 9 (800 mg, 81%) as an off-white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.3 Hz, 1H), 6.87 (d, J=2.8 Hz, 1H),6.76 (dd, J=8.3, 2.8 Hz, 1H), 4.12 (s, 2H), 4.11 (s, 2H), 4.01 (t, J=5.2Hz, 2H), 4.00-3.97 (m, 1H), 3.64 (dt, J=14.0, 5.1 Hz, 1H), 3.53-3.43 (m,1H), 3.22 (t, J=7.0 Hz, 2H), 2.31 (s, 3H), 2.30 (s, 3H), 1.74-1.54 (m,3H), 1.51 (s, 9H), 1.46 (s, 9H), 1.44-1.32 (3H), 1.41 (s, 9H).

Preparation of compound B:(R)-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)-6-guanidinohexanamideHydrochloride

A solution of 9 (800 mg, 1.02 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (171mg, 4.08 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (146 mg,0.51 mmol) and stirred for another 1 h. The organic solvent was removedand the residue was partitioned between saturated aqueous NaHCO₃solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated andthe aqueous layer was extracted with CH₂Cl₂ (2×30 mL). The combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated. Thefinal residue was dissolved in EtOH (20 mL) and 4 N HCl (20 mL) wasadded. After stirring at room temperature for 1 h, the reaction mixturewas concentrated to afford compound B (crude HCl salt) as a yellowsolid. The crude HCl salt was purified by reverse-phase columnchromatography and lyophilized to afford 310 mg (66%) of pure compound Bas a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.4Hz, 1H), 6.90 (d, J=2.5 Hz, 1H), 6.78 (dd, J=8.4, 2.5 Hz, 1H), 4.08 (t,J=5.1 Hz, 2H), 3.89 (t, J=7.0 Hz, 1H), 3.82 (s, 2H), 3.80 (s, 2H), 3.71(dt, J=14.0, 5.1 Hz, 1H), 3.61-3.63 (m, 1H), 3.08 (t, J=7.0 Hz, 2H),1.93-1.79 (m, 2H), 1.64-1.53 (m, 2H), 1.50-1.40 (m, 2H); ¹H NMR (400MHz, DMSO-d₆) δ 8.88 (t, J=5.7 Hz, 1H), 8.33 (br s, 3H), 7.90 (t, J=5.8Hz, 1H), 7.70-6.67 (m, 4H), 7.22 (d, J=8.5 Hz, 1H), 6.92 (d, J=2.6 Hz,1H), 6.79 (dd, J=8.5, 2.6 Hz, 1H), 4.07-3.96 (m, 2H), 3.83-3.74 (m, 5H),3.57-3.42 (m, 2H), 3.10-3.01 (m, 2H), 2.95 (t, J=7.4 Hz, 1H), 2.82 (t,J=7.0 Hz, 1H), 1.81-1.66 (m, 2H), 1.52-1.40 (m, 2H), 1.39-1.28 (m, 2H);HRMS (ESI-MS m/z) calculated for C₁₇H₂₉N₅O₂S₂ [M+H]⁺, 400.1841. found400.1855.

3. Preparation of C:(S)-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)-6-guanidinohexanamide

Preparation of Compound 11

Compound A (429 mg, 1.12 mmol) and acid 10 (550 mg, 1.22 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (0.78 mL, 4.48 mmol) andHATU (927 mg, 2.44 mmol). The reaction mixture was stirred at roomtemperature for 24 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and saturated aqueous NaHCO₃ (50 mL). The organic layer was separated,washed with brine (50 mL), and dried over Na₂SO₄. The organic layer wasconcentrated and the residue was purified by column chromatography(silica gel, 50% to 80% EtOAc in hexanes) to afford compound 11 (550 mg,63%) as a yellow oil: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.3 Hz, 1H),6.87 (d, J=2.8 Hz, 1H), 6.77 (dd, J=8.3, 2.8 Hz, 1H), 4.12 (s, 2H), 4.11(s, 2H), 4.01 (t, J=5.2 Hz, 2H), 4.00-3.97 (m, 1H), 3.64 (dt, J=14.0,5.1 Hz, 1H), 3.53-3.43 (m, 1H), 3.22 (t, J=7.0 Hz, 2H), 2.32 (s, 3H),2.30 (s, 3H), 1.74-1.54 (m, 3H), 1.51 (s, 9H), 1.46 (s, 9H), 1.44-1.32(3H), 1.41 (s, 9H).

Preparation of compound C; [ALB-167699(a)];(S)-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)-6-guanidinohexanamide

A solution of 11 (800 mg, 1.02 mmol) in a mixture of THF (6.0 mL),methanol (6.0 mL), and water (6.0 mL) was charged with solid LiOH.H₂O(129 mg, 3.06 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (146 mg, 0.51 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated to yield 600 mg of white, solid product. 400 mg of thecrude product was dissolved in EtOH (5.0 mL) and 4 N HCl (20 mL) wasadded. After stirring at room temperature for 1 h, the reaction mixturewas concentrated to afford compound C (crude HCl salt) as a yellowsolid. The crude HCl salt was purified by reverse-phase columnchromatography and lyophilized to afford 120 mg (45%) of pure compound Cas a hygroscopic yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.4Hz, 1H), 6.90 (d, J=2.5 Hz, 1H), 6.78 (dd, J=8.4, 2.5 Hz, 1H), 4.08 (t,J=5.1 Hz, 2H), 3.86 (t, J=6.5 Hz, 1H), 3.82 (s, 2H), 3.80 (s, 2H), 3.71(dt, J=14.0, 5.1 Hz, 1H), 3.61-3.63 (m, 1H), 3.07 (t, J=7.0 Hz, 2H),1.93-1.79 (m, 2H), 1.64-1.53 (m, 2H), 1.48-1.38 (m, 2H); ¹H NMR (400MHz, DMSO-d₆) δ 8.84 (t, J=5.7 Hz, 1H), 8.32 (br s, 3H), 7.82 (t, J=5.7Hz, 1H), 7.60-6.68 (m, 4H), 7.21 (d, J=8.5 Hz, 1H), 6.91 (d, J=2.6 Hz,1H), 6.78 (dd, J=8.5, 2.6 Hz, 1H), 4.06-3.98 (m, 2H), 3.82-3.72 (m, 5H),3.56-3.43 (m, 2H), 3.09-2.99 (m, 2H), 2.93 (t, J=7.4 Hz, 1H), 2.80 (t,J=7.0 Hz, 1H), 1.76-1.68 (m, 2H), 1.50-1.41 (m, 2H), 1.38-1.27 (m, 2H);HRMS (ESI-MS m/z) calculated for C₁₇H₂₉N₅O₂S₂ [M+H]⁺, 400.1835. found400.1855.

4. Preparation of D:(R)-2-amino-N—OR)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexyl)-6-guanidinohexanamideHydrochloride Preparation of(R)-6-[2,3-bis(tert-butoxycarbonyl)guanidino]-2-[(tert-butoxycarbonyl)amino]hexanoicacid (8)

A solution of N-α-Boc-D-lysine 12 (10.0 g, 40.6 mmol) in CH₂Cl₂ (200 mL)was charged with N,N′-bis-Boc-1-guanylpyrazole (11.3 g, 36.6 mmol) andtriethylamine (11.0 mL, 81.3 mmol). The reaction mixture was stirred atroom temperature for 16 h. The reaction mixture was washed with 10%aqueous citric acid (2×100 mL) and the solvent was removed under reducedpressure. The residue was dissolved in 1 N NaOH (300 mL), 1 N HCl wasadded to adjust the pH to 5-6, and the mixture was extracted with CH₂Cl₂(500 ml). The CH₂Cl₂ layer was separated and the aqueous layer wasextracted with CH₂Cl₂ (2×250 mL). The combined organic layers were driedover Na₂SO₄, filtered, and concentrated to afford compound 8 (18.5 g,94%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 4.13-4.03 (m, 1H), 3.36(t, J=6.8 Hz, 2H), 1.91-1.77 (m, 1H), 1.74-1.55 (m, 3H), 1.52 (s, 9H),1.51-1.38 (m, 2H), 1.47 (s, 9H), 1.43 (s, 9H).

Preparation of (R)-methyl6-amino-2-[(tert-butoxycarbonyl)amino]hexanoate (14)

A solution of N-α-Boc-D-lysine 12 (1.00 g, 4.06 mmol) in CH₂Cl₂ (20 mL)and MeOH (5.0 mL) was charged with TMS-diazomethane [(CH₃)₃SiCHN₂ 0.6 Msolution in hexane, 13.5 mL, 8.12 mmol] at 0° C. and stirred at the sametemperature for 1 h and at room temperature for another 1 h. The solventwas removed under reduced pressure to afford crude product 14 (1.04 g,crude) as a yellow oil, which was directly used for the next stepwithout further purification: ¹H NMR (400 MHz, CD₃OD) δ 4.12-4.05 (m,1H), 3.70 (s, 3H), 2.62 (t, J=7.0 Hz, 2H), 1.82-1.70 (m, 1H), 1.69-1.58(m, 1H), 1.54-1.32 (m, 4H), 1.43 (s, 9H).

Preparation of (12R,19R)-methyl6,12,19-tris[(tert-butoxycarbonyl)amino]-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazaicos-5-en-20-oate(15

A stirred solution of acid 8 (1.95 g, 4.00 mmol) and amine 14 (1.04 g,4.00 mmol) in CH₂Cl₂ (60 mL) was charged with NMM (2.64 mL, 24.0 mmol)and EEDQ (1.97 g, 8.00 mmol). The resulting mixture was stirred at roomtemperature for 16 h. Water (20 mL) was added to the reaction mixtureand extracted with CH₂Cl₂ (3×50 mL). The combined organic extract waswashed with brine and dried over Na₂SO₄. The solvent was removed and theresidue was purified by column chromatography (silica gel, 20% to 40%EtOAc in hexanes) to afford amide 15 (2.15 g, 73% over two steps) as awhite solid: ¹H NMR (400 MHz, CD₃OD) δ 4.10-4.02 (m, 1H), 4.01-3.92 (m,1H), 3.70 (s, 3H), 3.35 (t, J=6.8 Hz, 2H), 3.24-3.10 (m, 2H), 1.83-1.67(m, 2H), 1.66-1.55 (m, 4H), 1.52 (s, 9H), 1.46 (s, 9H), 1.45-1.35 (m,6H), 1.438 (s, 9H), 1.431 (s, 9H).

Preparation of(12R,19R)-6,12,19-tris[(tert-butoxycarbonyl)amino]-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazaicos-5-en-20-oicacid (16)

A solution of methyl ester 15 (2.15 g, 2.94 mmol) in MeOH/THF/H₂O (30mL/30 mL/15 mL) was charged with NaOH (589 mg, 14.7 mmol) and thereaction mixture was stirred at room temperature for 1 h. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure and the pH was adjusted to 5 with 1 N HCl. The suspension waspartitioned between CH₂Cl₂ (50 mL) and water (50 mL). The organic layerwas separated and the aqueous layer was extracted with CH₂Cl₂ (2×50 mL).The combined organic extracts were dried over Na₂SO₄ and concentrated toafford compound 16 (1.80 g, 86%) as a white solid, which was useddirectly in the next step: ¹H NMR (400 MHz, CD₃OD) δ 4.07-4.00 (m, 1H),4.00-3.91 (m, 1H), 3.35 (t, J=7.2 Hz, 2H), 3.25-3.10 (m, 2H), 1.89-1.23(m, 12H), 1.52 (s, 9H), 1.46 (s, 9H), 1.438 (s, 9H), 1.436 (s, 9H).

Preparation of Compound 17

Compound A (700 mg, 1.96 mmol) and acid 16 (1.40 g, 1.96 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (1.71 mL, 9.80 mmol) andHATU (745 mg, 1.96 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and saturated aqueous NaHCO₃ (50 mL). The organic layer was separated,washed with brine (50 mL), and dried over Na₂SO₄. The organic layer wasconcentrated and the residue was purified by column chromatography(silica gel, 50% to 80% EtOAc in hexanes) to afford compound 17 (1.30 g,66%) as a pink solid: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.4 Hz, 1H),6.87 (d, J=2.6 Hz, 1H), 6.76 (dd, J=8.4, 2.6 Hz, 1H), 4.12 (d, J=2.9 Hz,4H), 4.00 (t, J=5.5 Hz, 2H), 3.99-3.92 (m, 2H), 3.67-3.57 (m, 1H),3.56-3.45 (m, 1H), 3.34 (t, J=6.9 Hz, 2H), 3.20-3.01 (m, 2H), 2.33 (s,3H), 2.31 (s, 3H), 1.78-1.54 (m, 6H), 1.51 (s, 9H), 1.46 (s, 9H),1.45-1.27 (m, 6H), 1.43 (s, 9H), 1.40 (s, 9H).

Preparation of Compound D

A solution of 17 (1.30 g, 1.28 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (216mg, 5.14 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture TCEP.HCl (183 mg, 0.64 mmol) wascharged with and stirred for another 1 h. The solvent was removed andthe residue was partitioned between saturated aqueous NaHCO₃ solution(10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and theaqueous layer was extracted with CH₂Cl₂ (2×30 mL). The organic layerswere combined, dried over Na₂SO₄, filtered, and concentrated. Theresidue was dissolved in EtOH (20 mL) and 4 N HCl (20 mL) was added.After stirring at room temperature for 1 h, the reaction mixture wasconcentrated to afford compound D (820 mg, crude HCl salt) as a yellowsolid. The crude HCl salt (600 mg) was purified by reverse-phase columnchromatography and lyophilized to afford 325 mg (55%) of pure compound Das a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.4Hz, 1H), 6.91 (d, J=2.7 Hz, 1H), 6.78 (dd, J=8.4, 2.7 Hz, 1H), 4.14-4.04(m, 2H), 3.86 (td, J=6.7, 2.7 Hz, 2H), 3.83 (s, 2H), 3.80 (s, 2H),3.75-3.66 (m, 1H), 3.62-3.52 (m, 1H), 3.21 (t, J=7.5 Hz, 2H), 3.17-3.08(m, 2H), 1.96-1.75 (m, 4H), 1.71-1.59 (m, 2H), 1.58-1.34 (m, 6H); ¹H NMR(400 MHz, DMSO-d₆) δ 8.84 (t, J=6.0 Hz, 1H), 8.68 (t, J=5.6 Hz, 1H),8.43-8.15 (m, 5H), 7.84 (t, J=6.0 Hz, 1H), 7.65-6.61 (m, 4H), 7.22 (d,J=8.4 Hz, 1H), 6.91 (d, J=2.6 Hz, 1H), 6.78 (dd, J=8.4, 2.6 Hz, 1H),4.08-3.96 (m, 2H), 3.79 (s, 2H), 3.77 (s, 2H), 3.74 (t, J=6.2 Hz, 1H),3.60-3.40 (m, 2H), 3.16-3.01 (m, 4H), 2.98-2.75 (m, 2H), 1.80-1.62 (m,4H), 1.54-1.39 (m, 4H), 1.39-1.27 (m, 4H); HRMS (ESI-MS m/z) calculatedfor C₂₃H₄₁N₇O₃S₂ [M+H]⁺, 528.2791. found 528.2794; Elemental analysis: %calcd C, 43.36; H 6.96; N, 14.11. found C, 41.23; H, 7.19; N, 15.39.

5. Preparation of E:(S)-2-amino-N-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexyl)-6-guanidinohexanamideHydrochloride

Preparation of(S)-6-[2,3-bis(tert-butoxycarbonyl)guanidino]-2-[(tert-butoxycarbonyl)amino]hexanoicacid (10)

A solution of N-α-Boc-L-lysine 18 (5.00 g, 20.3 mmol) in CH₂Cl₂ (100 mL)was charged with N,N′-bis-Boc-1-guanylpyrazole (5.10 g, 16.6 mmol) andtriethylamine (5.54 mL, 40.6 mmol). The reaction mixture was stirred atroom temperature for 48 h. The reaction mixture was washed with 10%aqueous citric acid (2×80 mL) and the solvent was removed under reducedpressure. The residue was dissolved in 1 N NaOH (200 mL), the pH ofsolution was adjusted to 5-6 with 1 N HCl, and the mixture was extractedwith CH₂Cl₂ (300 ml). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×200 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated to afford compound 10(9.00 g, 91%) as a white solid: ¹H NMR (400 MHz, CD₃OD) δ 4.10-4.02 (m,1H), 3.36 (t, J=6.8 Hz, 2H), 1.90-1.78 (m, 1H), 1.73-1.56 (m, 3H), 1.52(s, 9H), 1.45-1.31 (m, 2H), 1.47 (s, 9H), 1.43 (s, 9H).

Preparation of (12S,19S)-methyl6,12,19-tris[(tert-butoxycarbonyl)amino]-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazaicos-5-en-20-oate(20)

A stirred solution of acid 10 (5.00 g, 10.2 mmol) and amine 19 (3.04 g,10.2 mmol) in CH₂Cl₂ (100 mL) was charged with NMM (6.75 mL, 61.5 mmol)and EEDQ (5.06 g, 20.5 mmol). The resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was charged with water (50mL) and extracted with CH₂Cl₂ (3×100 mL). The combined organic extractswere washed with brine and dried over Na₂SO₄. The solvent was removedand the residue was purified by column chromatography (silica gel, 20%to 40% EtOAc in hexanes) to afford amide 20 (6.00 g, 80%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 4.12-4.03 (m, 1H), 4.01-3.92 (m, 1H),3.70 (s, 3H), 3.35 (t, J=7.0 Hz, 2H), 3.25-3.11 (m, 2H), 1.83-1.69 (m,2H), 1.68-1.55 (m, 4H), 1.52 (s, 9H), 1.46 (s, 9H), 1.45-1.35 (m, 6H),1.438 (s, 9H), 1.436 (s, 9H).

Preparation of(12S,19S)-6,12,19-tris[(tert-butoxycarbonyl)amino]-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazaicos-5-en-20-oicacid, 21

A solution of methyl ester 20 (6.00 g, 8.21 mmol) in MeOH/THF/H₂O (100mL/100 mL/50 mL) was charged with NaOH (1.64 g, 41.1 mmol) and thereaction mixture was stirred at room temperature for 1 h. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure and the pH of the solution was adjusted to 5 with 1 N HCl. Thesuspension was partitioned between CH₂Cl₂ (150 mL) and water (100 mL).The organic layer was separated and the aqueous layer was extracted withCH₂Cl₂ (2×150 mL). The combined organic extracts were dried over Na₂SO₄and concentrated to afford compound 21 (5.50 g, 94%) as a white solid,which was used directly in the next step: ¹H NMR (400 MHz, CD₃OD) δ4.06-4.00 (m, 1H), 4.00-3.93 (m, 1H), 3.35 (t, J=7.3 Hz, 2H), 3.26-3.07(m, 2H), 1.85-1.56 (m, 6H), 1.53-1.32 (m, 6H), 1.52 (s, 9H), 1.46 (s,9H), 1.438 (s, 9H), 1.435 (s, 9H).

Preparation of Compound 22

Compound A (1.20 g, 3.42 mmol) and acid 21 (2.44 g, 3.42 mmol) weredissolved in DMF (25 mL) and treated with DIPEA (2.98 mL, 17.1 mmol) andHATU (1.30 g, 3.42 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was dissolved in CH₂Cl₂ (100 mL). Thesolution was quickly washed with saturated aqueous NaHCO₃ (2×50 mL) andbrine (50 mL) and dried over Na₂SO₄. The organic layer was concentratedand the residue was purified by column chromatography (silica gel, 50%to 80% EtOAc in hexanes) to afford compound 22 (1.50 g, 44%) as anorange solid: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.5 Hz, 1H), 6.87 (d,J=2.7 Hz, 1H), 6.76 (dd, J=8.5, 2.7 Hz, 1H), 4.12 (d, J=2.8 Hz, 4H),4.00 (t, J=5.4 Hz, 2H), 3.99-3.92 (m, 2H), 3.65-3.56 (m, 1H), 3.55-3.45(m, 1H), 3.34 (t, J=7.1 Hz, 2H), 3.21-3.04 (m, 2H), 2.33 (s, 3H), 2.31(s, 3H), 1.78-1.54 (m, 6H), 1.51 (s, 9H), 1.46 (s, 9H), 1.43-1.29 (m,6H), 1.43 (s, 9H), 1.40 (s, 9H).

Preparation of compound E:(S)-2-amino-N-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexyl)-6-guanidinohexanamideHydrochloride

A solution of 22 (1.50 g, 1.48 mmol) in a mixture of THF (10 mL),methanol (10 mL), and water (10 mL) was charged with solid LiOH.H₂O (249mg, 5.93 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (212 mg,0.74 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (5.0 mL) and 4 N HCl (20 mL) was added. After stirringat room temperature for 1 h, the reaction mixture was concentrated toafford crude compound 2048 as a yellow solid. The crude HCl salt (E) waspurified by reverse-phase column chromatography and lyophilized toafford 370 mg (39%) of pure compound E as a hygroscopic white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.4 Hz, 1H), 6.91 (d, J=2.7 Hz, 1H),6.78 (dd, J=8.4, 2.7 Hz, 1H), 4.14-4.04 (m, 2H), 3.87 (td, J=6.7, 2.7Hz, 2H), 3.83 (s, 2H), 3.80 (s, 2H), 3.75-3.66 (m, 1H), 3.62-3.52 (m,1H), 3.21 (t, J=7.5 Hz, 2H), 3.17-3.08 (m, 2H), 1.96-1.75 (m, 4H),1.71-1.59 (m, 2H), 1.58-1.34 (m, 6H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.86(t, J=5.6 Hz, 1H), 8.70 (t, J=5.6 Hz, 1H), 8.43-8.15 (m, 5H), 7.87 (t,J=5.7 Hz, 1H), 7.65-6.66 (m, 4H), 7.22 (d, J=8.4 Hz, 1H), 6.91 (d, J=2.6Hz, 1H), 6.78 (dd, J=8.4, 2.6 Hz, 1H), 4.08-3.96 (m, 2H), 3.79 (s, 2H),3.77 (s, 2H), 3.74 (t, J=6.2 Hz, 1H), 3.60-3.40 (m, 2H), 3.16-3.01 (m,4H), 2.98-2.75 (m, 2H), 1.80-1.62 (m, 4H), 1.54-1.39 (m, 4H), 1.39-1.27(m, 4H); HRMS (ESI-MS m/z) calculated for C₂₃H₄₁N₇O₃S₂ [M+H]⁺, 528.2791;found 528.2793.

6. Preparation of Branched Precursor F:S,S′-(4,5-bis(2-aminoethoxy)-1,2-phenylene)bis(methylene)diethanethioate Hydrochloride

Preparation of 5,6-dimethoxyisobenzofuran-1(3H)-one (24)

HCl gas was bubbled through an aqueous formaldehyde (37%, 70 mL) at 0°C. and then at room temperature to get a saturated solution (1.5 h).This solution was charged with 3,4-dimethoxybenzoic acid 23 (9.00 g,49.5 mmol) portionwise. The mixture was warmed to 70° C. and stirred atthat temperature for 7 h; HCl gas was continuously bubbled through thesolution during this period of time. The reaction mixture was stirred atroom temperature for 16 h. The solvent was removed, water (100 mL) wasadded, and the mixture was neutralized with aqueous NH₄OH solution. Asolid formed, which was filtered and washed with water.Recrystallization of the product from ethanol yielded a brown solid(5.00 g, 52%). 2.0 g of impure 24 was isolated as well.

Alternative Preparation of 24

Concentrated HCl (37%, 150 mL) was added to 3,4-dimethoxybenzoic acid 23(10.0 g, 54.9 mmol), followed by aqueous formaldehyde (37%, 75 mL). Themixture was warmed to 90° C. and stirred at that temperature for 5 h.The solvent was removed and the residue was portioned between water (100mL) and EtOAc (250 ml). The organic layer was separated and the aqueouslayer was extracted with EtOAc (3×200 mL). The combined organic layerwas washed with 2.5 M NaOH, followed by water, and concentrated. Theresidue was purified by column chromatography (silica gel, 25 to 50%EtOAc in hexanes) to afford compound 24 (7.00 g, 66%) as an off-whitesolid: ¹H NMR (400 MHz, DMSO-d₆) δ 7.26 (s, 1H), 7.23 (s, 1H), 5.27 (s,2H), 3.87 (s, 3H), 3.84 (s, 3H).

Preparation of 5,6-dihydroxyisobenzofuran-1(3H)-one (25)

A solution of compound 24 (7.00 g, 36.1 mmol) in CH₂Cl₂ (150 mL) wascooled to −78° C. and BBr₃ (8.52 mL, 90.2 mmol) was added at the sametemperature. Stirring was continued at −78° C. for 30 min, and thereaction mixture was brought to room temperature and stirred for 16 h.The reaction mixture was quenched with MeOH at 0° C. and the solvent wasremoved. The residue was portioned between water (100 mL) and EtOAc (200mL); the EtOAc layer was separated. The aqueous layer was extracted withEtOAc (3×200 mL). The combined organic layer was concentrated and theresidue was purified by column chromatography (silica gel, 40-60% EtOAcin hexanes) to afford compound 25 (5.00 g, 83%) as an off-white solid:¹H NMR (400 MHz, DMSO-d₆) δ 10.18 (br s, 1H), 9.65 (br s, 1H), 7.06 (s,1H), 6.92 (s, 1H), 5.16 (s, 2H).

Preparation of di-tert-butyl{[(1-oxo-1,3-dihydroisobenzofuran-5,6-diyl)bis(oxy)]bis(ethane-2,1-diyl)}dicarbamate(26)

A solution of compound 25 (5.00 g, 30.1 mmol) in DMF (40 mL) was chargedwith K₂CO₃ (16.6 g, 120 mmol) and stirred at room temperature for 5 min.The above reaction mixture was charged with compound 3 (21.1 g, 90.4mmol) and the reaction mixture was stirred at room temperature for 120h. The reaction mixture was diluted with water (300 mL) and extractedwith EtOAc (3×300 mL). The combined organic layer was concentrated andthe residue was purified by column chromatography (silica gel, 30-60%EtOAc in hexanes) to afford compound 26 (8.00 g, 59%) as a white gum: ¹HNMR (400 MHz, CD₃OD) δ 7.72 (s, 1H), 7.16 (s, 1H), 5.24 (s, 2H), 4.14(t, J=5.7 Hz, 2H), 4.08 (t, J=5.7 Hz, 2H), 3.54-3.44 (m, 4H), 1.43 (s,18H).

Preparation of di-tert-butyl({[4,5-bis(hydroxymethyl)-1,2-phenylene]bis(oxy)}bis(ethane-2,1-diyl))dicarbamate(27)

A solution of compound 26 (5.00 g, 11.0 mmol) in THF (50 mL) was chargedwith lithium aluminum hydride (1 M solution in diethyl ether, 33.2 mL,33.2 mmol) at 0° C. The resulting reaction mixture was stirred at 0° C.for 1 h and quenched with ice-cold water at 0° C. The reaction mixturewas diluted with chloroform (300 mL) and filtered through a Celite pad,and the Celite pad was washed with chloroform (2×300 ml). The filtratewas concentrated under vacuum to afford 27 (4.50 g, 90%) as a colorlessgum: ¹H NMR (400 MHz, DMSO-d₆) δ 6.99 (s, 2H), 6.90 (t, J=5.9 Hz, 2H),4.97 (brs, 2H), 4.44 (s, 4H), 3.92 (t, J=5.6 Hz, 4H), 3.30-3.22 (m, 4H),1.38 (s, 18H).

Preparation of 28

A solution of 27 (4.50 g, 9.95 mmol) in CH₂Cl₂ (100 mL) was charged withEt₃N (10.9 mL, 79.6 mmol) followed by methanesulfonyl chloride (3.00 mL,39.8 mmol) at 0° C. and stirred at room temperature for 12 h. Thereaction mixture was diluted with water (100 mL) and extracted withCH₂Cl₂ (3×150 mL). The combined organic extracts were washed with brine,dried over Na₂SO₄, and concentrated to afford the mesylated product(8.50 g, crude) as a yellow oil, which was directly used for the nextstep without further purification.

The above crude product (8.50 g, crude, 9.95 mmol) in a mixture of THF(200 ml) and DMF (50 mL) was charged with KSAc (2.84 g, 24.9 mmol) andstirred at room temperature for 16 h. The solvent was removed and theresidue was partitioned between water (50.0 mL) and CH₂Cl₂ (100 mL) TheCH₂Cl₂ layer was separated and the aqueous layer was extracted withCH₂Cl₂ (2×100 mL). The combined organic layer was concentrated and theresidue was purified by column chromatography (silica gel, 10% to 15%EtOAc in hexanes) to afford compound 28 (4.20 g, 74% over two steps) asa yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 6.91 (s, 2H), 4.10 (s, 4H),3.99 (t, J=5.7 Hz, 4H), 3.41 (t, J=5.6 Hz, 4H), 2.31 (s, 6H), 1.43 (s,18).

Preparation of compound F;S,S′-(4,5-bis(2-aminoethoxy)-1,2-phenylene)bis(methylene)diethanethioate Hydrochloride

Compound 28 (5.00 g, 8.74 mmol) was dissolved in 4 N HCl in dioxane (40mL) at room temperature and the solution was stirred for 2 h. Afterconcentration, the residue was triturated with MTBE to afford thehydrochloric acid salt F (3.50 g, 90%) as an off-white solid: ¹H NMR(400 MHz, CD₃OD) δ 7.02 (s, 2H), 4.24 (t, J=5.2 Hz, 4H), 4.13 (s, 4H),3.39 (t, J=5.3 Hz, 4H), 2.32 (s, 6H).

7. Preparation of G:(2R,2′R)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)Hydrochloride

Preparation of Compound 29

Compound F (888 mg, 2.00 mmol) and acid 8 (1.95 g, 4.00 mmol) weredissolved in DMF (20 mL) and treated with DIPEA (3.49 mL, 20.0 mmol) andHATU (1.52 g, 4.00 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. The solvent was removed under reducedpressure and the residue was partitioned between CH₂Cl₂ (100 mL) andsaturated aqueous NaHCO₃ (50 mL). The organic layer was separated,washed with brine (50 mL), and dried over Na₂SO₄. The organic layer wasconcentrated and the residue was purified by column chromatography(silica gel, 50% to 80% EtOAc in hexanes) to afford compound 29 (1.31 g,50%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 6.88 (s, 2H), 4.09 (s,4H), 4.08-3.98 (m, 6H), 3.69-3.53 (m, 4H), 3.23 (t, J=7.0 Hz, 4H), 2.31(s, 6H), 1.79-1.57 (m, 6H), 1.57-1.30 (m, 6H), 1.51 (s, 18H), 1.46 (s,18H), 1.40 (s, 18H).

Preparation of compound G:(2R,2′R)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)Hydrochloride

A solution of 29 (1.31 g, 1.00 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (168mg, 4.00 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (143 mg,0.50 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between CH₂Cl₂ (50 mL) and saturated aqueousNaHCO₃ solution (10 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (20 mL) and 4 N HCl (20 mL) was added. After stirringat room temperature for 1 h, the reaction mixture was concentrated toafford compound G (800 mg, crude HCl salt) as a yellow solid. The crudeHCl salt (600 mg) was purified by reverse-phase column chromatographyand lyophilized to afford 135 mg (23%) of pure compound G as ahygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 6.95 (s, 2H), 4.11(t, J=5.8 Hz, 4H), 3.96 (t, J=6.8 Hz, 2H), 3.79 (s, 4H), 3.71 (dt,J=13.7, 5.8 Hz, 2H), 3.61 (dt, J=13.6, 5.0 Hz, 2H), 3.11 (t, J=7.0 Hz,4H), 1.97-1.84 (m, 4H), 1.67-1.55 (m, 4H), 1.53-1.43 (m, 4H); ¹HNMR (400MHz, DMSO-d₆) δ 9.03 (t, J=5.7 Hz, 2H), 8.35 (br s, 6H), 7.90 (t, J=5.4Hz, 2H), 7.73-6.78 (m, 7H), 6.98 (s, 2H), 4.02 (t, J=5.8 Hz, 4H),3.91-3.81 (m, 2H), 3.75 (s, 2H), 3.74 (s, 2H), 3.59-3.41 (m, 4H),3.11-3.03 (m, 4H), 2.91 (t, J=7.7 Hz, 2H), 1.81-1.70 (m, 4H), 1.52-1.41(m, 4H), 1.40-1.30 (m, 4H); HRMS (ESI-MS m/z) calculated forC₂₆H₄₈N₁₀O₄S₂ [M+H]⁺, 629.3380. found 629.3377; Elemental analysis: %calcd C, 40.31; H, 6.77; N, 18.08. found C, 35.32; H, 7.40; N, 15.76.

8. Preparation of H:(2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)Hydrochloride Preparation of Compound 30

Compound B (888 mg, 2.00 mmol) and acid 10 (1.95 g, 4.00 mmol) weredissolved in DMF (20 mL) and treated with DIPEA (3.49 mL, 20.0 mmol) andHATU (1.52 g, 4.00 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and NaHCO₃ (50 mL). The organic layer was separated, washed with brine(50 mL), and dried over Na₂SO₄. The organic layer was concentrated andthe residue was purified by column chromatography (silica gel, 50% to80% EtOAc in hexanes) to afford compound 30 (1.51 g, 57%) as anoff-white solid: ¹H NMR (400 MHz, CD₃OD) δ 6.88 (s, 2H), 4.09 (s, 4H),4.07-3.98 (m, 6H), 3.69-3.52 (m, 4H), 3.23 (t, J=7.0 Hz, 4H), 2.31 (s,6H), 1.79-1.59 (m, 6H), 1.57-1.30 (m, 6H), 1.51 (s, 18H), 1.46 (s, 18H),1.40 (s, 18H).

Preparation of compound H;(2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)Hydrochloride

A solution of 30 (1.51 g, 1.14 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (192mg, 4.57 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (163 mg,0.57 mmol) and stirred for another 1 h. The solvent was removed, theresidue was dissolved in CH₂Cl₂ (50 mL), and the solution was washedwith saturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated to get 600 mg of crude, white, solid product. 400 mg of thecrude product was dissolved in EtOH (5.0 mL), and 4 N HCl (20 mL) wasadded. After stirring at room temperature for 1 h, the reaction mixturewas concentrated to afford compound H (crude HCl salt) as a yellowsolid. The crude HCl salt was purified by reverse-phase columnchromatography and lyophilized to afford 420 mg (48%) of pure compound Has a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 6.95 (s, 2H),4.11 (t, J=5.8 Hz, 4H), 3.98 (t, J=6.8 Hz, 2H), 3.78 (s, 4H), 3.71 (dt,J=13.7, 5.8 Hz, 2H), 3.61 (dt, J=13.6, 5.0 Hz, 2H), 3.11 (t, J=7.0 Hz,4H), 1.97-1.84 (m, 4H), 1.67-1.55 (m, 4H), 1.53-1.43 (m, 4H); ¹H NMR(400 MHz, DMSO-d₆) δ 8.98 (br s, 2H), 8.30 (br s, 6H), 7.83 (br s, 2H),7.62-6.74 (m, 7H), 6.97 (s, 2H), 4.02 (t, J=5.8 Hz, 4H), 3.84 (t, J=6.9Hz, 2H), 3.75 (br s, 4H), 3.57-3.41 (m, 4H), 3.11-3.02 (m, 4H),2.96-2.83 (m, 2H), 1.81-1.70 (m, 4H), 1.52-1.41 (m, 4H), 1.40-1.30 (m,4H); HRMS (ESI-MS m/z) calculated for C₂₆H₄₈N₁₀O₄S₂ [M+H]⁺, 629.3380.found 629.3378.

9. Preparation of compound I: tert-butyl2-(3,4-bis(mercaptomethyl)phenoxy)ethylcarbamate

Preparation of Compound 31

A solution of 7 (12.9 g, 32.3 mmol) in methanol (250 mL) was chargedwith NH₄OH solution (40 mL, 37% in water) and the reaction mixture wasstirred at room temperature for 74 h. Solvent was removed, the residuewas partitioned between CH₂Cl₂ (250 mL) and water (100 mL). The CH₂Cl₂layer was separated and the aqueous layer was extracted with CH₂Cl₂(2×50 mL). The combined organic layers were dried over Na₂SO₄, filtered,and concentrated to get crude product which was purified by columnchromatography (silica gel, 2% to 6% MeOH in CH₂Cl₂) to afford compound31 (7.00 g, 66%, 80% pure by ¹H NMR) as a white solid: ¹H NMR (400 MHz,CDCl₃) δ 7.04 (d, J=8.4 Hz, 1H), 6.79-6.72 (m, 1H), 6.71-6.65 (m, 1H),5.33-5.07 (m, 1H), 3.98 (br s, 2H), 3.68 (s, 4H), 3.51 (br s, 2H), 1.43(s, 9H).

Preparation of compound I: tert-butyl2-(3,4-bis(mercaptomethyl)phenoxy)ethylcarbamate

A solution of 31 (500 mg, 1.52 mmol) in a mixture of THF (5.0 mL),methanol (5.0 mL), and NaHCO₃ saturated solution (5.0 mL) was chargedwith TCEP.HCl (656 mg, 2.29 mmol) and stirred for 2 h. The solvent wasremoved and the residue was partitioned between saturated aqueous NaHCO₃solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated andthe aqueous layer was extracted with CH₂Cl₂ (2×30 mL). The combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated. Theresidue was purified by preparative HPLC and lyophilized to afford 240mg (48%) of pure compound P-2035 as a light yellow liquid: ¹H NMR (400MHz, CD₃OD) δ 7.17 (d, J=8.5 Hz, 1H), 6.88 (d, J=2.7 Hz, 1H), 6.76 (dd,J=8.5, 2.7 Hz, 1H), 3.98 (t, J=5.3 Hz, 2H), 3.81 (s, 2H), 3.80 (s, 2H),3.40 (t, J=5.6 Hz, 2H), 1.43 (s, 9H); ¹H NMR (400 MHz, DMSO-d₆) δ 7.19(d, J=8.4 Hz, 1H), 6.97 (t, J=5.3 Hz, 1H), 6.89 (d, J=2.7 Hz, 1H), 6.76(dd, J=8.4, 2.7 Hz, 1H), 3.92 (t, J=6.0 Hz, 2H), 3.78 (d, J=4.5 Hz, 2H),3.76 (d, J=4.5 Hz, 2H), 3.27 (q, J=5.8 Hz, 2H), 2.88 (t, J=7.5 Hz, 1H),2.77 (t, J=7.5 Hz, 1H), 1.38 (s, 9H); ESI (m/z) [C₁₅H₂₃NO₃S₂+Na]⁺352.

10. Preparation of J: (S)-tert-butyl6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexane-1,5-diyldicarbamate

Preparation of Compound 33

Compound A (1.96 g, 5.60 mmol) and acid 32 (1.94 g, 5.60 mmol) weredissolved in DMF (25 mL) and treated with DIPEA (4.89 mL, 28.0 mmol) andHATU (2.12 g, 5.60 mmol). The reaction mixture was stirred at roomtemperature for 24 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was dissolved in CH₂Cl₂(100 mL). Thesolution was quickly washed with saturated aqueous NaHCO₃ (2×25 mL) andbrine (25 mL) and dried over Na₂SO₄. The organic layer was concentratedand the residue was purified by column chromatography (silica gel, 50%to 80% EtOAc in hexanes) to afford compound 33 (3.0 g, 86%) as ancolorless oil: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.5 Hz, 1H), 6.87(d, J=2.9 Hz, 1H), 6.76 (dd, J=8.5, 2.9 Hz, 1H), 4.13 (s, 2H), 4.12 (s,2H), 3.99 (t, J=5.5 Hz, 2H), 3.98-3.95 (m, 1H), 3.61 (td, J=13.9, 4.9Hz, 1H), 3.55-3.45 (m, 1H), 2.96 (t, J=6.9 Hz, 2H), 2.33 (s, 3H), 2.31(s, 3H), 1.75-1.64 (m, 1H), 1.63-1.52 (m, 1H), 1.49-1.26 (m, 4H), 1.42(s, 9H), 1.40 (s, 9H).

Preparation of compound J: (S)-tert-butyl6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexane-1,5-diyldicarbamate

A solution of 33 (750 mg, 1.19 mmol) in a mixture of THF (5.0 mL),methanol (5.0 mL), and water (5.0 mL) was charged with solid LiOH.H₂O(151 mg, 3.50 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (170 mg, 0.59 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (50 mL) The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was purified by preparative HPLC andlyophilized to afford 374 mg (56%) of pure compound J as a light yellowsolid: ¹H NMR (400 MHz, CD₃OD) δ 7.18 (d, J=8.4 Hz, 1H), 6.88 (d, J=2.5Hz, 1H), 6.78 (dd, J=8.4, 2.5 Hz, 1H), 4.03 (t, J=5.3 Hz, 2H), 4.01-3.94(m, 1H), 3.81 (s, 2H), 3.80 (s, 2H), 3.62 (td, J=14.1, 5.1 Hz, 1H), 3.53(t, J=5.1 Hz, 1H), 2.96 (t, J=6.4 Hz, 2H), 1.75-1.63 (m, 1H), 1.63-1.51(m, 1H), 1.50-1.24 (m, 4H), 1.42 (s, 18H); ¹H NMR (400 MHz, DMSO-d₆) δ7.96 (t, J=5.3 Hz, 1H), 7.20 (d, J=8.4 Hz, 1H), 6.88 (d, J=2.7 Hz, 1H),6.76 (dd, J=8.4, 2.7 Hz, 1H), 6.71-6.68 (m, 1H), 3.94 (t, J=6.1 Hz, 2H),3.90-3.81 (m, 1H), 3.78 (d, J=4.9 Hz, 2H), 3.76 (d, J=4.4 Hz, 2H),3.81-3.66 (m, 3H), 3.49-3.31 (m, 2H), 2.86 (t, J=7.5 Hz, 1H), 2.87-2.81(m, 2H), 2.76 (t, J=7.3 Hz, 1H), 1.59-1.41 (m, 2H), 1.39-1.12 (m, 4H),1.36 (s, 18H); HRMS (ESI-MS m/z) calculated for C₂₆H₄₃N₃O₆S₂ [M+H]⁺,558.2672. found 558.2678.

11. Preparation of K:(S)-6-acetamido-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamide

Preparation of Compound 34

Compound 33 (1.00 g, 1.59 mmol) was dissolved in 4 N HCl in dioxane (10mL) at room temperature, and the solution was stirred at sametemperature for 1 h. After removal of the solvent, the residue wastriturated with ethyl acetate and n-hexane to afford hydrochloric acidsalt 34 (800 mg, 98%) as an off-white solid: ¹H NMR (400 MHz, CD₃OD) δ7.20 (d, J=8.5 Hz, 1H), 6.89 (d, J=2.6 Hz, 1H), 6.79 (dd, J=8.5, 2.6 Hz,1H), 4.14 (s, 2H), 4.13 (s, 2H), 4.06 (t, J=5.3 Hz, 2H), 3.90 (t, J=6.5Hz, 1H), 3.72-3.63 (m, 1H), 3.62-3.54 (m, 1H), 2.87 (t, J=8.0 Hz, 2H),2.33 (s, 3H), 2.31 (s, 3H), 1.94-1.81 (m, 2H), 1.74-1.63 (m, 2H),1.5-1.41 (m, 2H).

Preparation of Compound K:(S)-6-acetamido-2-amino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamide

A solution of 34 (800 mg, 1.55 mmol) in water (10 mL) was charged withsolid LiOH.H₂O (262 mg, 6.23 mmol) and the reaction mixture was stirredat room temperature for 1 h. The above reaction mixture was charged withTCEP.HCl (222 mg, 0.77 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (100 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×50 mL) Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was purified by preparative HPLC andlyophilized to afford 90 mg (12%) of pure compound K as a colorless gum:¹H NMR (400 MHz, CD₃OD) δ 7.18 (d, J=8.4 Hz, 1H), 6.89 (d, J=2.5 Hz,1H), 6.77 (dd, J=8.4, 2.5 Hz, 1H), 4.05 (t, J=5.4 Hz, 2H), 3.81 (s, 2H),3.80 (s, 2H), 3.63 (td, J=13.9, 5.1 Hz, 1H), 3.54 (td, J=14.1, 4.8 Hz,1H), 3.36 (t, J=6.8 Hz, 1H), 3.13-3.01 (m, 2H), 1.89 (s, 3H), 1.75-1.52(m, 2H), 1.51-1.41 (m, 2H), 1.39-1.22 (m, 2H); ¹H NMR (400 MHz, DMSO-d₆)δ 8.14 (t, J=5.7 Hz, 1H), 7.74 (t, J=5.3 Hz, 1H), 7.20 (d, J=8.4 Hz,1H), 6.90 (d, J=2.7 Hz, 1H), 6.77 (dd, J=8.4, 2.7 Hz, 1H), 3.97 (t,J=5.6 Hz, 2H), 3.78 (s, 2H), 3.77 (s, 2H), 3.49-3.39 (m, 2H), 3.22-3.16(m, 2H), 3.12-2.83 (m, 2H), 2.96 (q, J=6.5 Hz, 2H), 1.76 (s, 3H),1.62-1.50 (m, 1H), 1.45-1.20 (m, 5H); HRMS (ESI-MS m/z) calculated forC₁₈H₂₉N₃O₃S₂ [M+H]⁺, 400.1729. found 400.1708.

12. Preparation of L:(S)-2,6-diamino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamide

A solution of 33 (300 mg, 0.46 mmol) in a mixture of THF (5.0 mL),methanol (5.0 mL), and water (5.0 mL) was charged with solid LiOH.H₂O(59 mg, 1.40 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (66 mg, 0.23 mmol) and stirred for another 1 h. The solvent wasremoved and the residue was partitioned between saturated aqueous NaHCO₃solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated andthe aqueous layer was extracted with CH₂Cl₂ (2×20 mL). The combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated. Theresidue was dissolved in EtOH (5.0 mL) and 4 N HCl (10 mL) was added.After stirring at room temperature for 1 h, the reaction mixture wasconcentrated to afford crude HCl salt which was purified byreverse-phase column chromatography and lyophilized to afford 90 mg(46%) of pure compound Kas a hygroscopic yellow solid: H NMR (400 MHz,CD₃OD) δ 7.20 (d, J=8.6 Hz, 1H), 6.90 (d, J=2.8 Hz, 1H), 6.78 (dd,J=8.6, 2.8 Hz, 1H), 4.12-4.04 (m, 2H), 3.89 (t, J=6.4 Hz, 1H), 3.83 (s,2H), 3.81 (s, 2H), 3.70 (ddd, J=10.8, 5.8, 4.6 Hz, 1H), 3.58 (ddd,J=9.9, 6.2, 4.3 Hz, 1H), 2.83 (dd, J=8.8, 6.9 Hz, 2H), 1.94-1.79 (m,2H), 1.72-1.61 (m, 2H), 1.52-1.40 (m, 2H); ¹H NMR (400 MHz, DMSO-d₆) δ8.84 (t, J=5.6 Hz, 1H), 8.30 (br s, 3H), 8.01 (br s, 3H), 7.22 (d, J=8.6Hz, 1H), 6.91 (d, J=2.7 Hz, 1H), 6.78 (dd, J=8.6, 2.7 Hz, 1H), 4.07-3.97(m, 2H), 3.80 (s, 2H), 3.78 (s, 2H), 3.83-3.71 (m, 1H), 3.58-3.41 (m,2H), 3.01-2.90 (m, 1H), 2.86-2.78 (m, 1H), 2.76-2.64 (m, 2H), 1.80-1.67(m, 2H), 1.63-1.50 (m, 2H), 1.42-1.29 (m, 2H); ESI(m/z)[C₁₆H₂₇N₃O₂S₂H]⁺358.

13. Preparation of M: (S)-tert-butyl16-(3,4-bis(mercaptomethyl)phenoxy)-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazahexadecan-12-yl-6-ylidenedicarbamate

Preparation of compound M: (S)-tert-butyl16-(3,4-bis(mercaptomethyl)phenoxy)-2,2-dimethyl-4,13-dioxo-3-oxa-5,7,14-triazahexadecan-12-yl-6-ylidenedicarbamate

A solution of 11 (800 mg, 1.02 mmol) in a mixture of THF (6.0 mL),methanol (6.0 mL), and water (6.0 mL) was charged with solid LiOH.H₂O(129 mg, 3.06 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (146 mg, 0.51 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (50 mL) The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated to yield 600 mg of white, solid product. 200 mg of thecrude product was purified by reverse-phase column chromatography andlyophilized to afford 96 mg (42%) of pure compound P-2040 as ahygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.17 (d, J=8.3 Hz,1H), 6.88 (d, J=2.5 Hz, 1H), 6.76 (dd, J=8.3, 2.5 Hz, 1H), 4.03 (t,J=5.3 Hz, 2H), 4.02-3.98 (m, 1H), 3.81 (s, 2H), 3.79 (s, 2H), 3.64 (dt,J=14.2, 5.3 Hz, 1H), 3.54-3.44 (m, 1H), 3.24 (t, J=7.3 Hz, 2H),1.76-1.66 (m, 1H), 1.64-1.56 (m, 1H), 1.56-1.48 (m, 2H), 1.51 (s, 9H),1.46 (s, 9H), 1.44-1.31 (m, 2H), 1.41 (s, 9H); ¹H NMR (400 MHz, DMSO-d₆)δ 11.48 (br s, 1H), 8.23 (t, J=5.1 Hz, 1H), 7.99 (t, J=5.4 Hz, 1H), 7.19(d, J=8.5 Hz, 1H), 6.89 (d, J=2.6 Hz, 1H), 6.76 (dd, J=8.5, 2.6 Hz, 1H),6.81-6.72 (m, 1H), 3.95 (t, J=5.6 Hz, 2H), 3.91-3.85 (m, 1H), 3.78 (d,J=5.3 Hz, 2H), 3.76 (d, J=4.7 Hz, 2H), 3.51-3.32 (m, 3H), 3.26-3.15 (m,2H), 2.85 (t, J=7.6 Hz, 1H), 2.76 (t, J=7.4 Hz, 1H), 1.62-1.47 (m, 2H),1.46 (s, 9H), 1.43-1.31 (m, 1H), 1.38 (s, 9H), 1.36 (s, 9H), 1.29-1.19(m, 3H); ESI (m/z) [C₃₂H₅₃N₅O₈S₂+H]⁺ 700.

14. Preparation of N:(2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylazanediyl)dihexane-1,2,3,4,5-pentaolhydrochloride

Preparation of Compounds 36; SG-SJL-C-164

A solution of amine A (218 mg, 0.66 mmol) in methanol (10 mL) wascharged with triol 35 (334 mg, 1.24 mmol) and acetic acid (0.2 mL, 3.30mmol) successively and stirred at room temperature for 10 min. Sodiumcyanoborohydride (78.0 mg, 1.24 mmol) was added to the above reactionmixture and the resulting reaction mixture was stirred at roomtemperature for 16 h. Additional 35 (2.0 equiv), AcOH (5.0 equiv), andNaCNBH₃ (2.0 equiv) were charged and the mixture was stirred for 24 h.Above reaction mixture was charged with NaHCO₃ (554 mg, 6.60 mmol) inwater (5.0 ml) at 0° C. and stirred for 10 min., (Boc)₂O (288 mg, 1.32mmol) was then added and the reaction mixture was stirred for 5 min atthe same temperature, brought to room temperature, and stirred foranother 1 h. After the solvent was removed under reduced pressure, theresidue was dissolved in EtOAc (200 mL). The solution was quickly washedwith saturated aqueous NaHCO₃ (2×50 mL) and brine (50 mL) and dried overNa₂SO₄ and purified by reverse-phase chromatography using a C18 Goldcolumn to get pure 36 (255 mg, 47%) as a white solid; 100 mgcorresponding mono-sugar Boc protected product (37) was isolated aswell: ¹H NMR (400 MHz, CD₃OD) δ 7.46-7.39 (m, 4H), 7.31-7.25 (m, 6H),7.14 (d, J=8.4 Hz, 1H), 6.83 (d, J=2.4 Hz, 1H), 6.67 (dd, J=8.4, 2.5 Hz,1H), 5.45 (s, 2H), 4.21 (dd, J=10.8, 5.6 Hz, 2H), 4.10 (s, 2H), 4.08 (s,2H), 4.07-3.99 (m, 4H), 3.97-3.91 (m, 2H), 3.90-3.87 (m, 2H), 3.72 (dd,J=9.6, 2.1 Hz, 2H), 3.57 (t, J=10.1 Hz, 2H), 3.16-3.07 (m, 2H), 3.01(dd, J=13.6, 4.1 Hz, 2H), 2.90 (dd, J=12.9, 9.1 Hz, 2H), 2.30 (s, 3H),2.29 (s, 3H).

Preparation of compound N:(2R,2′R,3R,3′R,4R,4′R,5S,5'S)-6,6′-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylazanediyl)dihexane-1,2,3,4,5-pentaolhydrochloride

A solution of 36 (100 mg, 0.12 mmol) in EtOH (1.0 mL) was charged with 4N HCl (2.0 ml) and the reaction mixture was stirred at room temperaturefor 1 h. The solvent was removed and the residue was dissolved in water(5.0 mL) and charged with solid LiOH.H₂O (25.0 mg, 0.60 mmol) and thereaction mixture was stirred at room temperature for 1 h. The abovereaction mixture was charged with TCEP.HCl (29 mg, 0.12 mmol) andstirred for another 1 h. The pH of above reaction mixture was brought to2 by aqueous 4 N HCl and solvent was removed. The crude HCl salt waspurified by reverse-phase column chromatography and lyophilized toafford 45 mg (63%) of pure compound P-2041 as a hygroscopic off-whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 7.22 (d, J=8.5 Hz, 1H), 6.99 (d, J=2.5Hz, 1H), 6.87 (dd, J=8.5, 2.5 Hz, 1H), 4.42-4.32 (m, 2H), 4.25-4.15 (m,2H), 3.83 (s, 2H), 3.82 (s, 2H), 3.85-3.81 (m, 2H), 3.77 (dd, J=10.8,3.1 Hz, 2H), 3.73-3.61 (m, 7H), 3.58-3.42 (m, 4H), 3.80-3.79 (m, 1H); ¹HNMR (400 MHz, DMSO-d₆) δ 8.63-8.52 (m, 1H), 7.22 (d, J=8.5 Hz, 1H), 6.97(d, J=2.6 Hz, 1H), 6.85 (dd, J=8.5, 2.6 Hz, 1H), 5.52 (d, J=4.6 Hz, 1H),5.44 (d, J=5.1 Hz, 1H), 4.81 (d, J=6.6 Hz, 2H), 4.64-4.50 (m, 4H),4.46-4.39 (m, 2H), 4.37-4.29 (m, 2H), 4.12-3.99 (m, 2H), 3.80 (d, J=3.0Hz, 2H), 3.79 (d, J=3.0 Hz, 2H), 3.74-3.65 (m, 4H), 3.64-3.56 (m, 2H),3.55-3.37 (m, 10H), 2.89 (t, J=7.5 Hz, 1H), 2.81 (t, J=7.1 Hz, 1H); ESI(m/z) [C₂₂H₃₉NO₁₁S₂+H]⁺558.

15. Preparation of O:(2R,3R,4R,5S)-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)hexane-1,2,3,4,5-pentaolhydrochloride

Preparation of Compound 37

A solution of amine A (1.00 g, 2.85 mmol) in methanol (50 mL) wascharged with triol 35 (992 mg, 3.70 mmol) and acetic acid (0.85 mL, 14.3mmol) successively and stirred at room temperature for 10 min. Sodiumcyanoborohydride (233 mg, 3.70 mmol) was added to the above reactionmixture and the resulting reaction mixture was stirred at roomtemperature for 65 h. Above reaction mixture was charged with saturatedNaHCO₃ (50 mL) in water at 0° C. and stirred for 10 min., (Boc)₂O (1.24g, 5.70 mmol) was then added and the reaction mixture was stirred for 5min at the same temperature, brought to room temperature, and stirredfor another 1 h. After the solvent was removed under reduced pressure,the residue was dissolved in EtOAc (250 mL). The solution was quicklywashed with saturated aqueous NaHCO₃ (2×50 mL) and brine (50 mL) anddried over Na₂SO₄ and purified by column chromatography (silica gel, 40%to 80% EtOAc in hexanes) to afford compound 37 (1.15 g, 61%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 7.52-7.43 (m, 2H), 7.33-7.25 (m, 3H),7.16 (d, J=8.5 Hz, 1H), 6.84 (d, J=2.4 Hz, 1H), 6.71 (dd, J=8.5, 2.4 Hz,1H), 5.54 (s, 1H), 4.23 (dd, J=10.9, 5.6 Hz, 1H), 4.118 (s, 2H), 4.110(s, 2H), 4.10-4.03 (m, 3H), 3.93 (ddd, J=14.5, 9.6, 5.1 Hz, 1H),3.83-3.77 (m, 1H), 3.76-3.70 (m, 1H), 3.68 (t, J=5.5 Hz, 1H), 3.67 (t,J=10.5 Hz, 2H), 3.63-3.57 (m, 1H), 3.45-3.33 (m, 1H), 2.314 (s, 3H),2.311 (s, 3H), 1.42 (s, 9H).

Preparation of Compound O:(2R,3R,4R,5S)-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)hexane-1,2,3,4,5-pentaolhydrochloride

A solution of 37 (1.15 g, 1.72 mmol) in EtOH (5.0 mL) was charged with 4N HCl (20 ml) and the reaction mixture was stirred at room temperaturefor 1 h. The solvent was removed and the residue was dissolved in water(10 mL) and charged with solid LiOH.H₂O (1.00 g, 24.0 mmol) and thereaction mixture was stirred at room temperature for 1 h. The abovereaction mixture was charged with TCEP.HCl (246 mg, 0.86 mmol) andstirred for another 1 h. The pH of above reaction mixture was brought to2 by aqueous 4 N HCl and solvent was removed. The crude HCl salt waspurified by reverse-phase column chromatography and lyophilized toafford 310 mg (42%) of pure compound O as a hygroscopic white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.23 (d, J=8.7 Hz, 1H), 6.98 (d, J=2.7 Hz, 1H),6.85 (dd, J=8.7, 2.7 Hz, 1H), 4.28 (t, J=5.1 Hz, 2H), 4.11 (ddd, J=9.6,7.1, 4.6 Hz, 1H), 3.88 (dd, J=4.7, 1.1 Hz, 1H), 3.82 (s, 2H), 3.82 (s,2H), 3.77 (dd, J=9.9, 2.5 Hz, 1H), 3.72-3.68 (m, 2H), 3.67-3.62 (m, 1H),3.51-3.45 (m, 2H), 3.34-3.29 (m, 2H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.67(br s, 2H), 7.24 (d, J=8.5 Hz, 1H), 6.96 (d, J=2.6 Hz, 1H), 6.82 (dd,J=8.5, 2.6 Hz, 1H), 5.40 (d, J=4.6 Hz, 1H), 4.85-4.75 (m, 1H), 4.66-4.55(m, 1H), 4.60 (d, J=5.5 Hz, 1H), 4.44 (d, J=6.1 Hz, 1H), 4.23 (t, J=5.7Hz, 2H), 3.99-3.91 (m, 1H), 3.80 (d, J=7.6 Hz, 2H), 3.78 (d, J=7.1 Hz,2H), 3.73-3.67 (m, 1H), 3.63-3.56 (m, 1H), 3.54-3.38 (m, 3H), 3.34 (t,J=5.1 Hz, 2H), 3.20 (dd, J=13.2, 3.5 Hz, 1H), 3.06 (dd, J=12.4, 9.1 Hz,1H), 2.91 (t, J=8.0 Hz, 1H), 2.81 (t, J=7.5 Hz, 1H); ESI (m/z)[C₁₆H₂₇NO₆S₂+H]⁺ 394.

16. Preparation of P:(2R,3R,4R,5S)-6-((2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaolhydrochloride

Preparation of Compounds 38; SG-SJL-C-176

A solution of amine A (700 mg, 2.00 mmol) in methanol (40 mL) wascharged with triol (35) (697 mg, 2.60 mmol) and acetic acid (0.60 mL,10.0 mmol) successively and stirred at room temperature for 10 min.Sodium cyanoborohydride (164 mg, 2.60 mmol) was added and the finalreaction mixture was stirred at room temperature for 20 h. The abovereaction mixture was charged with hexanal (0.60 mL, 5.00 mmol), aceticacid (0.60 ml, 10.0 mL) followed by NaCNBH₃ (315 mg, 5.00 mmol), stirredfor 1 h. After the solvent was removed under reduced pressure, theresidue was dissolved in EtOAc (150 mL). The solution was quickly washedwith saturated aqueous NaHCO₃ (2×50 mL) and brine (50 mL) and dried overNa₂SO₄. Purification by normal chromatography using CMA system failed togive pure product, the mixture was then purified by reverse-phasechromatography using a C18 Gold column to get pure 38 (650 mg, 50%) as agum: ¹H NMR (400 MHz, CD₃OD) δ 7.48-7.42 (m, 2H), 7.33-7.27 (m, 3H),7.17 (d, J=8.5 Hz, 1H), 6.84 (d, J=2.4 Hz, 1H), 6.72 (dd, J=8.5, 2.4 Hz,1H), 5.49 (s, 1H), 4.23 (dd, J=10.6, 5.3 Hz, 1H), 4.112 (s, 2H), 4.110(s, 2H), 4.02-3.96 (m, 3H), 3.94 (dd, J=9.9, 5.1 Hz, 1H), 3.90 (dd,J=5.5, 2.2 Hz, 1H), 3.76 (dd, J=9.1, 2.2 Hz, 1H), 3.58 (t, J=10.1 Hz,2H), 3.52-3.40 (m, 1H), 2.96-2.84 (m, 2H), 2.69 (dd, J=12.3, 7.3 Hz,1H), 2.61 (dd, J=8.3, 5.9 Hz, 1H), 2.308 (s, 3H), 2.301 (s, 3H),1.50-1.39 (m, 2H), 1.31-1.18 (m, 6H), 0.86 (t, J=6.8 Hz, 3H).

Preparation of compound P:(2R,3R,4R,5S)-6-((2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)(hexyl)amino)hexane-1,2,3,4,5-pentaolhydrochloride

A solution of 38 (650 mg, 1.00 mmol) in EtOH (5.0 mL) was charged with 4N HCl (20 ml) and the reaction mixture was stirred at room temperaturefor 1 h. The solvent was removed and the residue was dissolved in water(10 mL) and charged with solid LiOH.H₂O (600 mg, 14.0 mmol) and thereaction mixture was stirred at room temperature for 1 h. The abovereaction mixture was charged with TCEP.HCl (143 mg, 0.50 mmol) andstirred for another 1 h. The pH of above reaction mixture was brought to2 by aqueous 4 N HCl and solvent was removed. The crude HCl salt waspurified by reverse-phase column chromatography and lyophilized toafford 230 mg (45%) of pure compound P as a hygroscopic off-white solid:¹H NMR (400 MHz, CD₃OD) δ 7.27 (d, J=8.6 Hz, 1H), 6.96 (d, J=2.6 Hz,1H), 6.87 (dd, J=8.6, 2.6 Hz, 1H), 4.38 (t, J=4.7 Hz, 2H), 4.24-4.14 (m,1H), 3.79 (s, 2H), 3.78 (s, 2H), 3.77-3.74 (m, 2H), 3.73-3.63 (m, 3H),3.63-3.56 (m, 2H), 3.49-3.36 (m, 2H), 3.30 (t, J=8.1 Hz, 2H), 1.80-1.64(m, 2H), 1.37-1.12 (m, 6H), 0.78 (t, J=6.6 Hz, 3H); ¹H NMR (400 MHz,DMSO-d₆) δ 9.58 (d, J=14.5 Hz, 1H), 7.25 (d, J=8.5 Hz, 1H), 6.96 (d,J=1.6 Hz, 1H), 6.83 (dd, J=8.5, 1.6 Hz, 1H), 5.51 (br s, 1H), 4.83 (brs, 1H), 4.60 (br s, 1H), 4.50-4.38 (m, 1H), 4.36 (br s, 1H), 4.14-4.01(m, 1H), 3.80 (d, J=5.6 Hz, 2H), 3.78 (d, J=5.6 Hz, 2H), 3.74-3.67 (m,1H), 3.66-3.53 (m, 3H), 3.52-3.45 (m, 2H), 3.42 (dd, J=10.6, 4.6 Hz,2H), 3.25-3.10 (m, 3H), 2.92 (t, J=7.6 Hz, 1H), 2.82 (t, J=6.8 Hz, 1H),1.76-1.63 (m, 2H), 1.35-1.21 (m, 6H), 0.87 (t, J=6.8 Hz, 3H); HRMS(ESI-MS m/z) calculated for C₂₂H₃₉NO₆S₂ [M+H]⁺, 478.2297; found478.2268.

17. Preparation of Q:(4-(2-(dimethylamino)ethoxy)-1,2-phenylene)dimethanethiol hydrochloride

Preparation of Compounds 39; SG-SJL-C-181

To a solution of compound A (700 mg, 2.00 mmol) and formaldehydesolution (30% in water, 1.20 mL, 12.0 mmol) in MeOH (20 mL) was addedAcOH (1.20 mL, 20.0 mmol) and the reaction mixture was stirred at roomtemperature for 10 min. After NaCNBH₃ (756 mg, 12.0 mmol) was added, thesolution was continued to be stirred at room temperature for 1 h. Afterremoval of solvent, the residue was neutralized with saturated NaHCO₃and the residue was partitioned between EtOAc (100 mL) and water (30mL). The EtOAc layer was separated and aqueous layer was extracted withEtOAc (2×40 mL) The combined organic extracts were dried over Na₂SO₄ andconcentrated under vacuum. The crude product 39 (700 mg) as a yellowliquid was directly used for the next step without any purification: ¹HNMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.7 Hz, 1H), 6.89 (d, J=2.5 Hz, 1H),6.78 (dd, J=8.7, 2.5 Hz, 1H), 4.13 (s, 2H), 4.12 (s, 2H), 4.09 (t, J=5.3Hz, 2H), 2.84 (t, J=5.2 Hz, 2H), 2.40 (s, 6H), 2.32 (s, 3H), 2.31 (s,3H).

Preparation of compound Q:(4-(2-(dimethylamino)ethoxy)-1,2-phenylene)dimethanethiol hydrochloride

A solution of 39 (700 mg, ˜2.00 mmol) in a mixture of THF (10 mL),methanol (10 mL), and water (10 mL) was charged with solid LiOH.H₂O (420mg, 10.0 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (572 mg,2.00 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×25 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The above crudeproduct was acidified with aqueous 4 N HCl and solvent was removed. Thecrude HCl salt was purified by reverse-phase column chromatography andlyophilized to afford 110 mg (19%, over two steps) of pure compound Q asa hygroscopic off-white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.25 (d, J=8.5Hz, 1H), 7.00 (d, J=2.8 Hz, 1H), 6.87 (dd, J=8.5, 2.5 Hz, 1H), 4.34 (t,J=4.9 Hz, 2H), 3.84 (s, 2H), 3.82 (s, 2H), 3.58 (t, J=5.1 Hz, 2H), 2.98(s, 6H); ¹H NMR (400 MHz, DMSO-d₆) δ 10.31 (br s, 1H), 7.25 (d, J=8.2Hz, 1H), 6.97 (d, J=2.8 Hz, 1H), 6.84 (dd, J=8.2, 2.8 Hz, 1H), 4.32 (t,J=5.2 Hz, 2H), 3.80 (d, J=7.2 Hz, 2H), 3.79 (d, J=6.6 Hz, 2H), 3.47 (t,J=5.4 Hz, 2H), 2.92 (t, J=7.4 Hz, 1H), 2.82 (t, J=7.6 Hz, 1H), 2.82 (s,6H); ESI (m/z) [C₁₂N₁₉NOS₂+H]⁺ 258.

18. Preparation of R:3,5-diamino-N-(N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)carbamimidoyl)-6-chloropyrazine-2-carboxamidehydrochloride

Preparation of Compounds 41; SG-SJL-C-184

A solution of amine salt A (700 mg, 2.00 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (40, 1.24 g,3.20 mmol) in EtOH (20 mL) was charged with DIPEA (2.84 mL, 16.0 mmol)at room temperature. The reaction mixture was heated at 60° C. in asealed tube for 2 h, cooled to room temperature, and concentrated invacuo. The residue was purified by column chromatography (silica gel,80:18:2 CHCl₃/CH₃OH/NH₄OH) followed by reverse phase column to affordguanidine 41 (250 mg, impure) as a yellow solid; which was directly usedfor the next step; ESI (m/z) [C₂₀H₂₄ClN₇O₄S₂+H]⁺ 526.

Preparation of compound R:3,5-diamino-N-(N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)carbamimidoyl)-6-chloropyrazine-2-carboxamidehydrochloride

A solution of 41 (250 mg, ˜0.47 mmol) in a mixture of THF (5.0 mL),methanol (5.0 mL), and water (5.0 mL) was charged with solid LiOH.H₂O(100 mg, 2.38 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (135 mg, 0.47 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×25 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The above crude product was acidified with 4 N HCl andsolvent was removed. The crude HCl salt was purified by reverse-phasecolumn chromatography and lyophilized to afford 65 mg (7.0%, over twosteps) of pure compound P-2045 as a hygroscopic yellow solid: ¹H NMR(400 MHz, CD₃OD) δ 7.22 (d, J=8.6 Hz, 1H), 6.96 (d, J=2.5 Hz, 1H), 6.83(dd, J=8.6, 2.5 Hz, 1H), 4.23 (t, J=5.1 Hz, 2H), 3.83 (s, 2H), 3.81 (s,2H), 3.74 (t, J=4.8 Hz, 2H); ¹H NMR (400 MHz, DMSO-d₆) δ 10.57 (br s,1H), 9.42 (br s, 1H), 9.16-8.77 (m, 2H), 7.43 (br s, 2H), 7.24 (d, J=8.5Hz, 1H), 6.95 (d, J=2.6 Hz, 1H), 6.82 (dd, J=8.5, 2.6 Hz, 1H), 4.16 (t,J=5.3 Hz, 2H), 3.80 (s, 2H), 3.79 (s, 2H), 3.74-3.66 (m, 2H), 2.95-2.86(m, 1H), 2.84-2.76 (m, 1H); ESI (m/z) [C₁₆H₂₀ClN₇O₂S₂+H]⁺ 442.

19. Preparation of S:(S)-2,6-diamino-N-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexyl)hexanamide

Preparation of (S)-methyl6-((S)-2,6-bis((tert-butoxycarbonyl)amino)hexanamido)-2-((tert-butoxycarbonyl)amino)hexanoate(42); SG-SJL-D-77

A stirred solution of acid 32 (10.0 g, 28.9 mmol) and amine 19 (8.57 g,28.9 mmol) in CH₂Cl₂ (200 mL) was charged with NMM (19.1 mL, 174 mmol)and EEDQ (14.3 g, 57.8 mmol). The resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was charged with water (50mL) and extracted with CH₂Cl₂ (3×100 mL) The combined organic extractswere washed with brine and dried over Na₂SO₄. The solvent was removedand the residue was purified by column chromatography (silica gel, 20%to 40% EtOAc in hexanes) to afford amide 42 (14.6 g, 86%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 4.11-4.03 (m, 1H), 4.00-3.89 (m, 1H),3.70 (s, 3H), 3.26-3.11 (m, 2H), 3.02 (t, J=6.6 Hz, 2H), 1.83-1.60 (m,4H), 1.57-1.23 (m, 8H), 1.439 (s, 9H), 1.432 (s, 9H), 1.42 (s, 9H).

Preparation of(S)-6-((S)-2,6-bis((tert-butoxycarbonyl)amino)hexanamido)-2-((tert-butoxycarbonyl)amino)hexanoicacid (43); SG-SJL-D-81

A solution of methyl ester 42 (14.6 g, 24.8 mmol) in MeOH/THF/H₂O (225mL/225 mL/75 mL) was charged with NaOH (3.96 g, 99.2 mmol) and thereaction mixture was stirred at room temperature for 1 h. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure and the pH of the solution was adjusted to 5 with 1 N HCl. Thesuspension was partitioned between CH₂Cl₂ (250 mL) and water (100 mL).The organic layer was separated and the aqueous layer was extracted withCH₂Cl₂ (2×250 mL). The combined organic extracts were dried over Na₂SO₄and concentrated to afford compound 43 (13.8 g, 97%) as a white solid,which was used directly in the next step: ¹H NMR (400 MHz, CD₃OD) δ4.09-4.00 (m, 1H), 3.98-3.90 (m, 1H), 3.27-3.10 (m, 2H), 3.02 (t, J=6.9Hz, 2H), 1.87-1.74 (m, 2H), 1.73-1.61 (m, 2H), 1.60-1.23 (m, 8H), 1.48(s, 18H), 1.42 (s, 9H).

Preparation of compound 44; SG-SJL-D-10

Compound A (1.20 g, 3.42 mmol) and acid 43 (1.97 g, 3.42 mmol) weredissolved in DMF (25 mL) and treated with DIPEA (2.98 mL, 17.1 mmol) andHATU (1.30 g, 3.42 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was dissolved in CH₂Cl₂ (100 mL). Thesolution was quickly washed with saturated aqueous NaHCO₃ (2×50 mL) andbrine (50 mL) and dried over Na₂SO₄. The organic layer was concentratedand the residue was purified by column chromatography (silica gel, 50%to 80% EtOAc in hexanes) to afford compound 44 (2.00 g, 67%) as anyellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.4 Hz, 1H), 6.87 (d,J=2.6 Hz, 1H), 6.77 (dd, J=8.4, 2.6 Hz, 1H), 4.13 (s, 2H), 4.12 (s, 2H),4.00 (t, J=5.8 Hz, 2H), 3.97-3.88 (m, 2H), 3.66-3.56 (m, 1H), 3.55-3.45(m, 1H), 3.21-3.06 (m, 2H), 3.05-2.98 (m, 2H), 2.33 (s, 3H), 2.31 (s,3H), 1.77-1.62 (m, 2H), 1.62-1.53 (m, 2H), 1.53-1.25 (m, 8H), 1.43 (s,9H), 1.42 (s, 9H), 1.40 (s, 9H).

Preparation of compound(S)-2,6-diamino-N-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexyl)hexanamideS

A solution of 44 (1.25 g, 1.43 mmol) in a mixture of THF (10 mL),methanol (10 mL), and water (10 mL) was charged with solid LiOH.H₂O (242mg, 5.75 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (205 mg,0.71 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (5.0 mL) and 4 N HCl (20 mL) was added. After stiflingat room temperature for 1 h, the reaction mixture was concentrated toafford crude compound S as a yellow solid. The crude HCl salt (S) waspurified by reverse-phase column chromatography and lyophilized toafford 510 mg (60%) of pure compound S as a hygroscopic off-white solid:NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.5 Hz, 1H), 6.91 (d, J=2.7 Hz, 1H),6.78 (dd, J=8.5, 2.7 Hz, 1H), 4.12-4.05 (m, 2H), 3.90 (q, J=6.6 Hz, 2H),3.83 (s, 2H), 3.80 (s, 2H), 3.70 (ddd, J=10.5, 5.8, 4.6 Hz, 1H), 3.57(ddd, J=10.4, 6.0, 4.6 Hz, 1H), 3.21 (dt, J=13.5, 6.5 Hz, 1H), 3.12 (dt,J=13.9, 6.8 Hz, 1H), 2.96 (t, J=7.6 Hz, 2H), 1.99-1.79 (m, 4H),1.77-1.67 (m, 2H), 1.62-1.37 (m, 6H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.90(t, J=5.8 Hz, 1H), 8.76 (t, J=5.3 Hz, 1H), 8.55-7.92 (m, 10H), 7.22 (d,J=8.5 Hz, 1H), 6.92 (d, J=2.6 Hz, 1H), 6.78 (dd, J=8.5, 2.6 Hz, 1H),4.09-3.99 (m, 2H), 3.79 (s, 2H), 3.78 (s, 2H), 3.78-3.24 (m, 2H),3.55-3.41 (m, 2H), 3.06 (dd, J=12.8, 6.6 Hz, 2H), 3.01-2.91 (m, 1H),2.90-2.79 (m, 1H), 2.75 (t, J=7.5 Hz, 2H), 1.80-1.68 (m, 4H), 1.65-1.54(m, 2H), 1.49-1.29 (m, 6H); HRMS (ESI-MS m/z) calculated forC₂₂H₃₉N₅O₃S₂ [M+H]⁺, 486.2573. found 486.2559; Elemental analysis: %calcd C, 44.4; H, 7.11; N, 11.77. found C, 43.84; H, 6.66; N, 11.16.

20. Preparation of T:(S)-2,6-diamino-N-((S)-5-amino-6-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexylamino)-6-oxohexyl)hexanamide

Preparation of (10S,17S,24S)-methyl10,17,24-tris((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,18-trioxo-3-oxa-5,12,19-triazapentacosan-25-oate(45)

A stirred solution of acid 43 (1.50 g, 2.60 mmol) and amine 19 (775 mg,2.60 mmol) in CH₂Cl₂ (30 mL) was charged with NMM (1.71 mL, 15.6 mmol)and EEDQ (1.28 g, 5.20 mmol). The resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was charged with water (20mL) and extracted with CH₂Cl₂ (3×40 mL). The combined organic extractswere washed with brine and dried over Na₂SO₄. The solvent was removedand the residue was purified by column chromatography (silica gel, 40%to 80% EtOAc in hexanes) to afford amide 45 (2.00 g, 84%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) 4.10-4.02 (m, 1H), 3.98-3.88 (m, 2H),3.70 (s, 3H), 3.26-3.10 (m, 4H), 3.06-3.00 (m, 2H), 1.82-1.63 (m, 4H),1.62-1.26 (m, 14H), 1.439 (s, 18H), 1.431 (s, 9H), 1.42 (s, 9H).

Preparation of(10S,17S,24S)-10,17,24-tris((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,11,18-trioxo-3-oxa-5,12,19-triazapentacosan-25-oicacid (46)

A solution of methyl ester 45 (2.00 g, 2.18 mmol) in MeOH/THF/H₂O (60mL/60 mL/20 mL) was charged with NaOH (436 mg, 10.9 mmol) and thereaction mixture was stirred at room temperature for 1 h. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure and the pH of the solution was adjusted to 5 with 1 N HCl. Thesuspension was partitioned between CH₂Cl₂ (50 mL) and water (10 mL). Theorganic layer was separated and the aqueous layer was extracted withCH₂Cl₂ (2×50 mL). The combined organic extracts were dried over Na₂SO₄and concentrated to afford compound 46 (1.90 g, 97%) as a white solid,which was used directly in the next step: ¹H NMR (400 MHz, CD₃OD) δ4.08-4.00 (m, 1H), 3.98-3.89 (m, 2H), 3.27-3.10 (m, 4H), 3.02 (t, J=6.6Hz, 2H), 1.86-1.65 (m, 4H), 1.64-1.24 (m, 14H), 1.43 (s, 18H), 1.42 (s,18H).

Preparation of Compound 47

Compound A (349 mg, 1.00 mmol) and acid 46 (902 mg, 1.00 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (0.69 mL, 4.00 mmol) andHATU (380 mg, 1.00 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After solvent was removed under reducedpressure, the residue was dissolved in CH₂Cl₂ (40 mL). The solution wasquickly washed with saturated aqueous NaHCO₃ (2×25 mL) and brine (25 mL)and dried over Na₂SO₄. The organic layer was concentrated and theresidue was purified by column chromatography (silica gel, 50% to 100%EtOAc in hexanes) to afford compound 47 (700 mg, 64%) as an yellowsolid: ¹H NMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.5 Hz, 1H), 6.87 (d, J=2.7Hz, 1H), 6.77 (dd, J=8.5, 2.7 Hz, 1H), 4.13 (s, 2H), 4.12 (s, 2H), 4.00(t, J=5.8 Hz, 2H), 3.98-3.90 (m, 2H), 3.65-3.56 (m, 1H), 3.55-3.45 (m,1H), 3.27-3.06 (m, 5H), 3.02 (t, J=7.0 Hz, 2H), 2.33 (s, 3H), 2.31 (s,3H), 1.76-1.64 (m, 2H), 1.61-1.53 (m, 3H), 1.53-1.45 (m, 3H), 1.44-1.25(m, 10H), 1.434 (s, 9H), 1.431 (s, 9H), 1.42 (s, 9H), 1.40 (s, 9H).

Preparation of compound T:(S)-2,6-diamino-N-((S)-5-amino-6-((S)-5-amino-6-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylamino)-6-oxohexylamino)-6-oxohexyl)hexanamide

A solution of 10 (700 mg, 0.63 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (107mg, 2.55 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (90.0 mg,0.31 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (20 mL) and 4 N HCl (20 mL) was added. After stirringat room temperature for 1 h, the reaction mixture was concentrated toafford crude compound T as a yellow solid. The crude HCl salt (T) waspurified by reverse-phase column chromatography and lyophilized toafford 320 mg (67%) of pure compound Tas a hygroscopic white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.4 Hz, 1H), 6.91 (d, J=2.9 Hz, 1H),6.78 (dd, J=8.4, 2.9 Hz, 1H), 4.14-4.05 (m, 2H), 3.92 (t, J=7.0 Hz, 1H),3.91-3.85 (m, 2H), 3.83 (s, 2H), 3.81 (s, 2H), 3.75-3.67 (m, 1H),3.60-3.53 (m, 1H), 3.27-3.08 (m, 4H), 2.96 (dd, J=8.2, 7.8 Hz, 2H),1.99-1.79 (m, 6H), 1.78-1.68 (m, 2H), 1.65-1.37 (m, 10H); ¹H NMR (400MHz, DMSO-d₆) δ 8.89 (t, J=5.2 Hz, 1H), 8.77 (dd, J=12.3, 5.8 Hz, 2H),8.32 (br s, 8H), 8.10 (br s, 3H), 7.21 (d, J=8.6 Hz, 1H), 6.92 (d, J=2.4Hz, 1H), 6.78 (dd, J=8.6, 2.4 Hz, 1H), 4.09-3.97 (m, 2H), 3.85-3.71 (m,7H), 3.57-3.41 (m, 2H), 3.14-3.01 (m, 4H), 2.95 (t, J=8.0 Hz, 1H), 2.82(t, J=7.7 Hz, 1H), 2.80-2.68 (m, 2H), 1.79-1.68 (m, 6H), 1.67-1.53 (m,2H), 1.49-1.29 (m, 10H); HRMS (ESI-MS m/z) calculated for C₂₈H₅₁N₇O₄S₂[M+H]⁺, 614.3522. found 614.3530; Elemental analysis: % calcd C, 41.27;H 7.3; N, 12.91. found C, 41.83; H, 7.62; N, 12.22.

21. Preparation of U:(S)-2-amino-6-((S)-2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamido)-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamideHydrochloride

Preparation of (12S,19S,26S)-methyl6,12,19,26-tetrakis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,13,20-trioxo-3-oxa-5,7,14,21-tetraazaheptacos-5-en-27-oate(48)

A stirred solution of acid 21 (1.90 g, 2.60 mmol) and amine 19 (775 mg,2.60 mmol) in CH₂Cl₂ (30 mL) was charged with NMM (1.71 mL, 15.6 mmol)and EEDQ (1.28 g, 5.20 mmol). The resulting mixture was stirred at roomtemperature for 16 h. The reaction mixture was charged with water (20mL) and extracted with CH₂Cl₂ (3×40 mL). The combined organic extractswere washed with brine and dried over Na₂SO₄. The solvent was removedand the residue was purified by column chromatography (silica gel, 40%to 80% EtOAc in hexanes) to afford amide 48 (1.40 g, 51%) as a whitesolid: ¹H NMR (400 MHz, CD₃OD) 4.11-4.02 (m, 1H), 4.01-3.86 (m, 2H),3.70 (s, 3H), 3.35 (t, J=7.1 Hz, 2H), 3.24-3.12 (m, 4H), 1.81-1.67 (m,3H), 1.66-1.54 (m, 5H), 1.53-1.30 (m, 10H), 1.52 (s, 9H), 1.46 (s, 9H),1.438 (s, 9H), 1.437 (s, 9H), 1.432 (s, 9H).

Preparation of(12S,19S,26S)-6,12,19,26-tetrakis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,13,20-trioxo-3-oxa-5,7,14,21-tetraazaheptacos-5-en-27-oicacid (49)

A solution of methyl ester 48 (1.40 g, 1.32 mmol) in MeOH/THF/H₂O (45mL/45 mL/15 mL) was charged with NaOH (265 mg, 6.61 mmol) and thereaction mixture was stirred at room temperature for 1 h. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure and the pH of the solution was adjusted to 5 with aqueous 1 NHCl. The suspension was partitioned between CH₂Cl₂ (50 mL) and water (10mL). The organic layer was separated and the aqueous layer was extractedwith CH₂Cl₂ (2×50 mL) The combined organic extracts were dried overNa₂SO₄ and concentrated to afford compound 49 (1.20 g, 87%) as a whitesolid, which was used directly in the next step: ¹H NMR (400 MHz, CD₃OD)δ 4.06-3.89 (m, 3H), 3.35 (t, J=7.6 Hz, 2H), 3.25-3.11 (m, 4H),1.86-1.66 (m, 3H), 1.64-1.55 (m, 5H), 1.54-1.30 (m, 10H), 1.52 (s, 9H),1.46 (s, 9H), 1.43 (s, 27H).

Preparation of Compound 50

Compound A (200 mg, 0.57 mmol) and acid 49 (600 mg, 0.57 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (0.39 mL, 2.28 mmol) andHATU (216 mg, 0.57 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was dissolved in CH₂Cl₂ (40 mL). Thesolution was quickly washed with saturated aqueous NaHCO₃ (2×25 mL) andbrine (25 mL) and dried over Na₂SO₄. The organic layer was concentratedand the residue was purified by column chromatography (silica gel, 50%to 100% EtOAc in hexanes) to afford compound 50 (550 mg, 78%) as anbrown oil: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.5 Hz, 1H), 6.87 (d,J=2.7 Hz, 1H), 6.76 (dd, J=8.5, 2.7 Hz, 1H), 4.13 (s, 2H), 4.12 (s, 2H),4.00 (t, J=5.8 Hz, 2H), 3.98-3.88 (m, 2H), 3.66-3.56 (m, 1H), 3.55-3.46(m, 1H), 3.34 (t, J=7.0 Hz, 2H), 3.20-3.04 (m, 5H), 2.33 (s, 3H), 2.31(s, 3H), 1.79-1.65 (m, 3H), 1.64-1.54 (m, 5H), 1.53-1.33 (m, 10H), 1.51(s, 9H), 1.46 (s, 9H), 1.434 (s, 9H), 1.430 (s, 9H), 1.40 (s, 9H).

Preparation of compound U:(S)-2-amino-6-((S)-2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamido)-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamideHydrochloride

A solution of 50 (550 mg, 0.44 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O(75.0 mg, 1.76 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (63.0 mg, 0.22 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between saturated aqueousNaHCO₃ solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL) Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was dissolved in EtOH (20 mL) and 4 N HCl (20mL) was added. After stirring at room temperature for 1 h, the reactionmixture was concentrated to afford crude compound Uas a yellow solid.The crude HCl salt (U) was purified by reverse-phase columnchromatography and lyophilized to afford 210 mg (60%) of pure compoundP-2056 as a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.20 (d,J=8.6 Hz, 1H), 6.92 (d, J=2.6 Hz, 1H), 6.78 (dd, J=8.6, 2.6 Hz, 1H),4.13-4.05 (m, 2H), 3.89 (t, J=6.9 Hz, 1H), 3.87-3.84 (m, 2H), 3.83 (s,2H), 3.81 (s, 2H), 3.74-3.67 (m, 1H), 3.61-3.53 (m, 1H), 3.28-3.10 (m,4H), 3.22 (t, J=6.8 Hz, 2H), 1.98-1.76 (m, 6H), 1.70-1.39 (m, 12H); ¹HNMR (400 MHz, DMSO-d₆) δ 8.83 (t, J=5.3 Hz, 1H), 8.77-8.68 (m, 2H),8.39-8.19 (m, 8H), 7.83 (t, J=5.1 Hz, 1H), 7.22 (d, J=8.6 Hz, 1H), 6.91(d, J=2.4 Hz, 1H), 6.78 (dd, J=8.6, 2.4 Hz, 1H), 4.07-3.98 (m, 2H), 3.78(s, 2H), 3.77 (s, 2H), 3.83-3.69 (m, 3H), 3.55-3.45 (m, 2H), 3.15-3.01(m, 6H), 2.92 (t, J=7.5 Hz, 1H), 2.81 (t, J=7.3 Hz, 1H), 1.78-1.66 (m,6H), 1.53-1.39 (m, 6H), 1.35-1.26 (m, 6H); HRMS (ESI-MS m/z) calculatedfor C₂₉H₅₃N₉O₄S₂ [M+H]⁺, 656.3740. found 656.3770; Elemental analysis: %calcd C, 43.44; H, 7.17, N 15.72. found C, 39.51; H, 7.17; N, 14.23.

22. Preparation of V:(R)-2,6-diamino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamideHydrochloride

Preparation of Compound 52

Compound A (500 mg, 1.40 mmol) and acid 51 (486 mg, 1.40 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (0.98 mL, 5.60 mmol) andHATU (532 mg, 1.40 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was dissolved in CH₂Cl₂ (40 mL). Thesolution was quickly washed with saturated aqueous NaHCO₃ (2×25 mL) andbrine (25 mL) and dried over Na₂SO₄. The organic layer was concentratedand the residue was purified by column chromatography (silica gel, 50%to 100% EtOAc in hexanes) to afford compound 52 (640 mg, 71%) as anbrown oil: ¹H NMR (400 MHz, CD₃OD) δ 7.19 (d, J=8.4 Hz, 1H), 6.87 (d,J=2.7 Hz, 1H), 6.76 (dd, J=8.4, 2.7 Hz, 1H), 4.13 (s, 2H), 4.12 (s, 2H),4.00 (t, J=5.1 Hz, 2H), 3.98-3.93 (m, 1H), 3.61 (td, J=14.9, 5.5 Hz,1H), 3.55-3.45 (m, 1H), 2.96 (t, J=6.5 Hz, 2H), 2.33 (s, 3H), 2.31 (s,3H), 1.75-1.51 (m, 2H), 1.49-1.43 (m, 4H), 1.42 (s, 9H), 1.40 (s, 9H).

Preparation of Compound V:(R)-2,6-diamino-N-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)hexanamideHydrochloride

A solution of 52 (640 mg, 1.00 mmol) in a mixture of THF (10 mL),methanol (10 mL), and water (10 mL) was charged with solid LiOH.H₂O (168mg, 4.00 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (143 mg,0.50 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (10 mL) and 4 N HCl (10 mL) was added. After stirringat room temperature for 1 h, the reaction mixture was concentrated toafford crude compound P-2059 as a yellow solid. The crude HCl salt (V)was purified by reverse-phase column chromatography and lyophilized toafford 230 mg (54%) of pure compound V as a hygroscopic white solid: ¹HNMR (400 MHz, CD₃OD) δ 7.20 (d, J=8.5 Hz, 1H), 6.90 (d, J=2.7 Hz, 1H),6.78 (dd, J=8.5, 2.7 Hz, 1H), 4.12-4.04 (m, 2H), 3.89 (t, J=6.4 Hz, 1H),3.83 (s, 2H), 3.81 (s, 2H), 3.70 (ddd, J=10.8, 5.8, 4.6 Hz, 1H), 3.58(ddd, J=9.9, 6.2, 4.3 Hz, 1H), 2.83 (dd, J=8.8, 6.9 Hz, 2H), 1.94-1.79(m, 2H), 1.72-1.61 (m, 2H), 1.52-1.40 (m, 2H); ¹H NMR (400 MHz, DMSO-d₆)δ 8.90 (t, J=5.2 Hz, 1H), 8.25 (br s, 5H), 7.22 (d, J=8.6 Hz, 1H), 6.92(d, J=2.7 Hz, 1H), 6.78 (dd, J=8.6, 2.7 Hz, 1H), 4.07-3.97 (m, 2H), 3.80(s, 2H), 3.78 (s, 2H), 3.79-3.75 (m, 1H), 3.58-3.41 (m, 2H), 3.12-2.73(m, 2H), 2.69 (t, J=7.7 Hz, 2H), 1.81-1.67 (m, 2H), 1.63-1.50 (m, 2H),1.41-1.29 (m, 2H); HRMS (ESI-MS m/z) calculated for C₁₆H₂₇N₃O₂S₂ [M+H]⁺,358.1623. found 358.1612; Elemental analysis: % calcd C, 44.64; H 6.79;N, 9.76. found C, 42.85; H, 6.06; N, 9.17.

23. Preparation of W:(2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2,6-diaminohexanamide)Hydrochloride

Preparation of Compound 53

Compound F (888 mg, 2.00 mmol) and acid 32 (1.38 g, 4.00 mmol) weredissolved in DMF (20 mL) and treated with DIPEA (3.49 mL, 20.0 mmol) andHATU (1.52 g, 4.00 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and NaHCO₃ (50 mL). The organic layer was separated, washed with brine(50 mL), and dried over Na₂SO₄. The organic layer was concentrated andthe residue was purified by column chromatography (silica gel, 50% to80% EtOAc in hexanes) to afford compound 53 (1.50 g, 73%) as anoff-white solid: ¹H NMR (400 MHz, CD₃OD) δ 6.88 (s, 2H), 4.10 (s, 4H),4.09-3.96 (m, 6H), 3.63-3.55 (m, 4H), 2.97 (t, J=6.3 Hz, 4H), 2.31 (s,6H), 1.78-1.67 (m, 2H), 1.65-1.55 (m, 2H), 1.50-1.25 (m, 8H), 1.42 (s,18H), 1.40 (s, 18H).

Preparation of Compound W:(2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2,6-diaminohexanamide)Hydrochloride

A solution of 53 (1.50 g, 1.45 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (245mg, 5.83 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (207 mg,0.73 mmol) and stirred for another 1 h. The solvent was removed, theresidue was dissolved in CH₂Cl₂ (50 mL), and the solution was washedwith saturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was dissolved in EtOH (5.0 mL), and 4 N HCl(20 mL) was added. After stirring at room temperature for 1 h, thereaction mixture was concentrated to afford compound W (crude HCl salt)as a yellow solid. The crude HCl salt was purified by reverse-phasecolumn chromatography and lyophilized to afford 460 mg (46%) of purecompound P-W as a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) 6.96(s, 2H), 4.11 (t, J=5.8 Hz, 4H), 3.96 (t, J=6.6 Hz, 2H), 3.79 (s, 4H),3.71 (td, J=11.3, 5.8 Hz, 2H), 3.61 (td, J=11.3, 5.8 Hz, 2H), 2.86 (dd,J=8.9, 7.6 Hz, 4H), 1.97-1.84 (m, 4H), 1.73-1.63 (m, 4H), 1.54-1.44 (m,4H); ¹H NMR (400 MHz, DMSO-d₆) δ 9.00 (t, J=5.5 Hz, 2H), 8.66-7.57 (m,9H), 6.97 (s, 2H), 4.02 (t, J=6.0 Hz, 4H), 3.85 (t, J=6.5 Hz, 2H), 3.75(s, 4H), 3.59-3.49 (m, 2H), 3.45-3.39 (m, 2H), 2.69 (t, J=7.8 Hz, 4H),1.81-1.70 (m, 4H), 1.62-1.50 (m, 4H), 1.43-1.33 (m, 4H); HRMS (ESI-MSm/z) calculated for C₂₄H₄₄N₆O₄S₂ [M+H]⁺, 545.2944. found 545.2940;Elemental analysis: % calcd C, 41.74; H, 7.01; N, 12.17. found C, 37.86;H, 7.32; N, 11.08.

24. Preparation of X:(S,2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2,6-diaminohexanamido)hexanamide)

Preparation of Compound 54

Compound B (308 mg, 0.69 mmol) and acid 32 (800 mg, 1.39 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (1.20 mL, 6.90 mmol) andHATU (529 mg, 1.39 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and NaHCO₃ (50 mL). The organic layer was separated, washed with brine(50 mL), and dried over Na₂SO₄. The organic layer was concentrated andthe residue was purified by column chromatography (silica gel, 50% to80% EtOAc in hexanes) to afford compound 54 (700 mg, 73%) as a yellowsolid: ¹H NMR (400 MHz, CD₃OD) δ 6.89 (s, 2H), 4.10 (s, 4H), 4.08-3.99(m, 5H), 3.98-3.89 (m, 2H), 3.65-3.53 (m, 4H), 3.21-3.07 (m, 5H), 3.02(t, J=7.0 Hz, 4H), 2.32 (s, 6H), 1.78-1.53 (m, 9H), 1.52-1.27 (m, 15H),1.43 (s, 18H), 1.42 (s, 18), 1.40 (s, 18H).

Preparation of compound X:(S,2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2,6-diaminohexanamido)hexanamide)

A solution of 54 (700 mg, 0.50 mmol) in a mixture of THF (15 mL),methanol (15 mL), and water (15 mL) was charged with solid LiOH.H₂O(84.0 mg, 2.0 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (72 mg, 0.25 mmol) and stirred for another 1 h. The solvent wasremoved, the residue was dissolved in CH₂Cl₂ (50 mL), and the solutionwas washed with saturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂layer was separated and the aqueous layer was extracted with CH₂Cl₂(2×30 mL). The combined organic layers were dried over Na₂SO₄, filtered,and concentrated. The residue was dissolved in EtOH (15 mL), and 4 N HCl(15 mL) was added. After stirring at room temperature for 1 h, thereaction mixture was concentrated to afford compound P-2060 (crude HClsalt) as a yellow solid. The crude HCl salt was purified byreverse-phase column chromatography and lyophilized to afford 245 mg(48%) of pure compound P-2060 as a hygroscopic white solid: ¹H NMR (400MHz, CD₃OD) δ 6.96 (s, 2H), 4.12 (t, J=5.5 Hz, 4H), 3.98 (t, J=6.5 Hz,2H), 3.92 (t, J=6.5 Hz, 2H), 3.79 (s, 4H), 3.71 (td, J=14.5, 5.5 Hz,2H), 3.61 (td, J=14.1, 5.1 Hz, 2H), 3.22 (td, J=14.7, 7.1 Hz, 2H), 3.12(td, J=12.3, 6.4 Hz, 2H), 2.96 (t, J=7.4 Hz, 4H), 1.97-1.79 (m, 8H),1.78-1.68 (m, 4H), 1.61-1.41 (m, 12H); ¹H NMR (400 MHz, DMSO-d₆) δ 9.04(t, J=5.4 Hz, 2H), 8.76 (t, J=5.1 Hz, 2H), 8.44-8.23 (m, 12H), 8.07 (brs, 6H), 6.98 (s, 2H), 4.02 (t, J=5.3 Hz, 4H), 3.91-3.82 (m, 2H),3.82-3.76 (m, 2H), 3.76 (s, 2H), 3.74 (s, 2H), 3.59-3.40 (m, 5H),3.11-3.00 (m, 4H), 2.91 (t, J=7.2 Hz, 2H), 2.83-2.71 (m, 4H), 1.85-1.70(m, 8H), 1.67-1.54 (m, 4H), 1.50-1.28 (m, 12H); HRMS (ESI-MS m/z)calculated for C₃₆H₆₈N₁₀O₆S₂ [M+H]⁺, 801.4843; found 801.4799. Elementalanalysis: % calcd C, 42.4; H, 7.31; N, 13.73. found C, 39.42; H, 7.37;N, 12.55.

25. Preparation of Y:(S,2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamide)

Preparation of Compound 55

Compound F (444 mg, 1.00 mmol) and acid 21 (1.43 g, 2.00 mmol) weredissolved in DMF (10 mL) and treated with DIPEA (1.74 mL, 10.0 mmol) andHATU (760 mg, 2.00 mmol). The reaction mixture was stirred at roomtemperature for 16 h. TLC analysis of the yellow reaction mixture showedthe completion of the reaction. After the solvent was removed underreduced pressure, the residue was partitioned between CH₂Cl₂ (100 mL)and NaHCO₃ (50 mL) The organic layer was separated, washed with brine(50 mL), and dried over Na₂SO₄. The organic layer was concentrated andthe residue was purified by column chromatography (silica gel, 50% to80% EtOAc in hexanes) to afford compound 55 (810 mg, 48%) as an yellowsolid: ¹H NMR (400 MHz, CD₃OD) δ 6.89 (s, 2H), 4.10 (s, 4H), 4.07-3.94(m, 8H), 3.63-3.54 (m, 4H), 3.34 (t, J=7.3 Hz, 4H), 3.20-3.06 (m, 4H),2.31 (s, 6H), 1.79-1.65 (m, 4H), 1.64-1.54 (m, 10H), 1.50-1.30 (m, 10H),1.51 (s, 18H), 1.46 (s, 18H), 1.43 (s, 18H), 1.39 (s, 18H).

Preparation of compound Y:(S,2S,2′S)-N,N′-(2,2′-(4,5-bis(mercaptomethyl)-1,2-phenylene)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamide)

A solution of 55 (810 mg, 0.48 mmol) in a mixture of THF (15 mL),methanol (15 mL), and water (15 mL) was charged with solid LiOH.H₂O (81mg, 1.92 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (69 mg,0.24 mmol) and stirred for another 1 h. The solvent was removed, theresidue was dissolved in CH₂Cl₂ (50 mL), and the solution was washedwith saturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was dissolved in EtOH (15 mL), and 4 N HCl (15mL) was added. After stirring at room temperature for 1 h, the reactionmixture was concentrated to afford compound Y (crude HCl salt) as ayellow solid. The crude HCl salt was purified by reverse-phase columnchromatography and lyophilized to afford 290 mg (55%) of pure compound Yas a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 6.96 (s, 2H),4.11 (t, J=5.3 Hz, 4H), 3.95 (t, J=6.5 Hz, 2H), 3.87 (t, J=6.8 Hz, 2H),3.79 (s, 4H), 3.74-3.66 (m, 2H), 3.65-3.57 (m, 2H), 3.21 (t, J=8.9, 7.2Hz, 6H), 3.18-3.08 (m, 2H), 1.94-1.78 (m, 8H), 1.69-1.60 (m, 4H),1.59-1.40 (m, 12H); ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (t, J=5.8 Hz, 2H),8.75 (t, J=5.5 Hz, 2H), 8.70-8.02 (m, 9H), 7.91 (t, J=4.8 Hz, 2H),7.77-6.76 (m, 8H), 6.98 (s, 2H), 4.02 (t, J=5.8 Hz, 4H), 3.85 (t, J=6.7Hz, 2H), 3.76 (t, J=6.5 Hz, 2H), 3.75 (s, 4H), 3.58-3.41 (m, 8H),3.16-3.08 (m, 4H), 3.08-3.01 (m, 4H), 1.83-1.68 (m, 8H), 1.54-1.28 (m,16H); HRMS (ESI-MS m/z) calculated for C₃₈H₇₂N₁₄O₆S₂ [M+H]⁺, 885.5279.found 885.5304; Elemental analysis: % calcd C, 41.34; H, 7.12; N, 17.76.found C, 39.04; H, 7.32; N, 15.89.

26. Preparation of Z:(2R,2′R)-N,N′-(3,3′-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)

Preparation of Compound 57

Compound A (356 mg, 1.00 mmol) and NaBH(AcO)₃ (530 mg, 2.50 mmol) weredissolved in 1,2-DCE (20 mL) and stirred at room temperature for 5 min.Known aldehyde 56 (1.36 g, 2.50 mmol) was added and the reaction mixturewas stirred at room temperature for 3 h. TLC analysis (100% EtOAc) ofthe reaction mixture showed incomplete reaction. The reaction mixturewas charged with NaHCO₃ solution (25 mL) and extracted with CH₂Cl₂ (3×50mL). The organic layer was concentrated and the residue was purified bycolumn chromatography (silica gel, 4% to 6% MeOH in EtOAc to CH₂Cl₂) toafford compound 57 (1.00 g, mixture) as a white solid, mixture wasdirectly used for the next step: ¹H NMR (400 MHz, CD₃OD) δ 7.21 (d,J=8.8 Hz, 1H), 6.91 (d, J=2.7 Hz, 1H), 6.80 (dd, J=8.8, 2.7 Hz, 1H),4.13 (s, 2H), 4.12 (s, 2H), 4.05 (t, J=6.0 Hz, 2H), 4.01-3.93 (m, 2H),3.28-3.23 (m, 4H), 3.38-3.32 (m, 4H), 2.70-2.65 (m, 2H), 2.64-2.52 (m,4H), 2.33 (s, 3H), 2.31 (s, 3H), 1.77-1.66 (m, 6H), 1.65-1.30 (m, 6H),1.47-1.32 (m, 4H), 1.51 (s, 18H), 1.46 (s, 18H), 1.42 (s, 18H).

Preparation of compound Z:(2R,2′R)-N,N′-(3,3′-(2-(3,4-bis(mercaptomethyl)phenoxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)

A solution of 57 (1.00 g, 0.73 mmol, mixture) in a mixture of THF (20mL), methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O(123 mg, 2.92 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (105 mg, 0.36 mmol) and stirred for another 1 h. The solventwas removed and the residue was partitioned between CH₂Cl₂ (50 mL) andsaturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was dissolved in EtOH (20 mL) and 4 N HCl (20mL) was added. After stirring at room temperature for 1 h, the reactionmixture was concentrated to afford compound Z (640 mg, crude HCl salt)as a yellow solid. The crude HCl salt (490 mg) was purified byreverse-phase column chromatography and lyophilized to afford 40 mg (8%,over two steps) of pure compound Zas a hygroscopic white solid: ¹H NMR(400 MHz, CD₃OD) δ 7.25 (d, J=8.4 Hz, 1H), 7.03 (d, J=2.6 Hz, 1H), 6.90(dd, J=8.4, 2.6 Hz, 1H), 4.41 (t, J=5.0 Hz, 2H), 3.92 (t, J=6.5 Hz, 2H),3.85 (s, 2H), 3.82 (s, 2H), 3.73-3.66 (m, 2H), 3.51-3.36 (m, 6H), 3.33(t, J=7.2 Hz, 2H), 3.22 (t, J=7.7 Hz, 4H), 2.17-2.03 (m, 4H), 1.99-1.79(m, 4H), 1.69-1.59 (m, 4H), 1.55-1.43 (m, 4H); ¹H NMR (400 MHz, DMSO-d₆)δ 10.75 (br s, 1H), 8.99-8.91 (m, 2H), 8.44-8.26 (m, 5H), 7.81 (t, J=6.0Hz, 2H), 7.69-6.77 (m, 7H), 7.25 (d, J=8.6 Hz, 1H), 7.01 (d, J=2.4 Hz,1H), 6.87 (dd, J=8.6, 2.4 Hz, 1H), 4.40 (t, J=5.1 Hz, 2H), 3.80 (s, 2H),3.77 (s, 2H), 3.84-3.72 (m, 2H), 3.58-3.50 (m, 2H), 3.28-3.17 (m, 8H),3.15-3.07 (m, 4H), 2.99 (t, J=7.7 Hz, 1H). 2.83 (t, J=7.3 Hz, 1H),2.03-1.87 (m, 4H), 1.78-1.68 (m, 4H), 1.54-1.42 (m, 4H), 1.39-1.29 (m,4H); HRMS (ESI-MS m/z) calculated for C₃₀H₅₇N₁₁O₃S₂ [M+H]⁺, 684.4166.found 684.4169.

27. Preparation of AA:(S)-2,6-diamino-N-(2-(1,4-dihydrobenzo[d][1,2]dithiin-6-yloxy)ethyl)hexanamide

A solution of 33 (320 mg, 0.49 mmol) in a mixture of THF (10 mL),methanol (10 mL), and water (10 mL) was charged with solid LiOH.H₂O (105mg, 2.49 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (128 mg,0.45 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between saturated aqueous NaHCO₃ solution (10mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×20 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated to get 300 mg crudeproduct as a white solid. 300 mg crude product was dissolved in EtOH(5.0 mL) and 4 N HCl (15 mL) was added. After stirring at roomtemperature for 1 h, the reaction mixture was concentrated to affordcrude HCl salt (280 mg, crude) which was dissolved in methanol (30 mL)and stirred at room temperature for 48 h in open air (<10% formation ofdisulfide was observed by LCMS analyses). To the above reaction mixtureNH₄OH (30% in water, 20 mL) was added and stirred at room temperaturefor another 72 h in open air (˜50% formation of disulfide was observedby LCMS analyses). Then DMSO (1.0 mL) was added to the above reactionmixture and stirring was continued for more 48 h (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 29 mg(18%, three steps) of pure compound AA as a hygroscopic brownsemi-solid: ¹H NMR (400 MHz, CD₃OD) δ 7.04 (d, J=8.4 Hz, 1H), 6.78 (dd,J=8.4, 2.6 Hz, 1H), 6.72 (d, J=2.6 Hz, 1H), 4.10-4.04 (m, 2H), 4.03 (s,2H), 4.00 (s, 2H), 3.78 (t, J=6.8 Hz, 1H), 3.68 (ddd, J=10.4, 5.8, 4.6Hz, 1H), 3.57 (ddd, J=10.9, 6.4, 4.3 Hz, 1H), 2.83 (dd, J=7.5, 1.5 Hz,2H), 1.92-1.75 (m, 2H), 1.72-1.60 (m, 2H), 1.53-1.36 (m, 2H); ¹H NMR(400 MHz, DMSO-d₆) δ 8.63 (t, J=5.8 Hz, 1H), 8.40-7.27 (m, 4H), 7.08 (d,J=8.5 Hz, 1H), 6.80 (dd, J=8.5, 2.7 Hz, 1H), 6.75 (d, J=2.7 Hz, 1H),4.08 (s, 2H), 4.05 (s, 2H), 4.03-3.96 (m, 2H), 3.61 (t, J=6.6 Hz, 1H),3.56-3.33 (m, 2H), 2.70 (t, J=7.3 Hz, 2H), 1.71-1.60 (m, 2H), 1.57-1.48(m, 2H), 1.38-1.27 (m, 2H); ESI (m/z) [C₁₆H₂₅N₃O₂S₂+H]⁺356.

28. Preparation of BB:(2S,2′S)-N,N′-(2,2′-(1,4-dihydrobenzo[d][1,2]dithiine-6,7-diyl)bis(oxy)bis(ethane-2,1-diyl))bis(2,6-diaminohexanamide)

A solution of W (65 mg, 0.09 mmol) in methanol (5.0 mL), was chargedwith NH₄OH (30% in water, 3.0 mL) and DMSO (0.5 mL) and reaction mixturewas stirred at room temperature for 96 h in open air (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 49 mg(96%) of pure compound BB as a hygroscopic white solid: ¹H NMR (400 MHz,CD₃OD) δ 6.79 (s, 2H), 4.09 (t, J=5.3 Hz, 4H), 3.98 (s, 4H), 3.95 (t,J=6.6 Hz, 2H), 3.70 (td, J=13.9, 5.5 Hz, 2H), 3.66 (td, J=14.2, 4.8 Hz,2H), 2.86 (ddd, J=7.5, 6.4, 1.3 Hz, 4H), 2.00-1.81 (m, 4H), 1.75-1.61(m, 4H), 1.56-1.41 (m, 4H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.99 (t, J=4.8Hz, 2H), 8.40-8.19 (m, 4H), 8.11-7.82 (m, 4H), 6.82 (s, 2H), 4.02 (s,4H), 4.06-3.95 (m, 5H), 3.90-3.80 (m, 2H), 3.74-3.18 (m, 3H), 2.82-2.62(m, 4H), 1.83-1.68 (m, 4H), 1.63-1.48 (m, 4H), 1.45-1.25 (m, 4H); HRMS(ESI-MS m/z) calculated for C₂₄H₄₂N₆O₄S₂ [M+H]⁺, 543.2787; found543.2779; Elemental analysis: % calcd C, 53.11; H, 7.8; N, 15.48. foundC, 37.21; H, 6.79; N, 11.02.

29. Preparation of CC:(2S,2′S)-N,N′-(2,2′-(1,4-dihydrobenzo[d][1,2]dithiine-6,7-diyl)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)

A solution of H (55 mg, 0.09 mmol) in methanol (5.0 mL), was chargedwith NH₄OH (30% in water, 3.0 mL) and DMSO (0.5 mL) and reaction mixturewas stirred at room temperature for 96 h in open air (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 15 mg(28%) of pure compound CC as a hygroscopic white solid: ¹H NMR (400 MHz,CD₃OD) δ 6.77 (s, 2H), 4.08 (t, J=5.5 Hz, 4H), 3.97 (s, 4H), 3.84 (t,J=7.1 Hz, 2H), 3.68 (dt, 13.9, 4.8 Hz, 2H), 3.60 (dt, J=13.9, 5.3 Hz,2H), 3.08 (t, J=7.0 Hz, 4H), 1.93-1.74 (m, 4H), 1.64-1.52 (m, 4H),1.49-1.38 (m, 4H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (t, J=5.5 Hz, 2H),8.40-8.20 (m, 5H), 7.78 (t, J=6.2 Hz, 2H), 7.67-6.81 (m, 7H), 6.82 (s,2H), 4.01 (s, 4H), 4.09-3.93 (m, 4H), 3.90-3.77 (m, 2H), 3.58-3.40 (m,4H), 3.08-3.00 (m, 4H), 1.81-1.66 (m, 4H), 1.52-1.41 (m, 4H), 1.40-1.25(m, 4H); HRMS (ESI-MS m/z) calculated for C₂₆H₄₆N₁₀O₄S₂ [M+H]⁺,627.3223. found 627.3260

30. Preparation of DD:(2R,2′R)-N,N′-(2,2′-(1,4-dihydrobenzo[d][1,2]dithiine-6,7-diyl)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-guanidinohexanamide)

A solution of 29 (1.31 g, 1.00 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (168mg, 4.00 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (143 mg,0.50 mmol) and stirred for another 1 h. The solvent was removed and theresidue was partitioned between CH₂Cl₂ (50 mL) and saturated aqueousNaHCO₃ solution (10 mL). The CH₂Cl₂ layer was separated and the aqueouslayer was extracted with CH₂Cl₂ (2×30 mL). The combined organic layerswere dried over Na₂SO₄, filtered, and concentrated. The residue wasdissolved in EtOH (20 mL) and 4 N HCl (20 mL) was added. After stirringat room temperature for 1 h, the reaction mixture was concentrated toafford compound G (800 mg, crude HCl salt) as a yellow solid. A solutionof G (200 mg, 0.25 mmol, crude) in methanol (10 mL), was charged withNH₄OH (30% in water, 10 mL) and DMSO (0.5 mL) and reaction mixture wasstirred at room temperature for 20 h in open air (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 56 mg(36%, over two steps) of pure compound P-DD as a hygroscopic whitesolid: ¹H NMR (400 MHz, CD₃OD) δ 6.78 (s, 2H), 4.09 (t, J=5.5 Hz, 4H),3.98 (s, 4H), 3.98 (t, J=7.3 Hz, 2H), 3.72 (dt, J=13.9, 4.8 Hz, 2H),3.59 (dt, J=13.9, 5.3 Hz, 2H), 3.08 (t, J=7.0 Hz, 4H), 1.97-1.81 (m,4H), 1.65-1.54 (m, 4H), 1.53-1.41 (m, 4H); ¹H NMR (400 MHz, DMSO-d₆) δ900 (t, J=5.8 Hz, 2H), 8.31 (br s, 5H), 7.85 (t, J=5.8 Hz, 2H),7.70-6.68 (m, 7H), 6.83 (s, 2H), 4.01 (s, 4H), 4.05-3.97 (m, 4H),3.89-3.81 (m, 2H), 3.58-3.40 (m, 4H), 3.10-3.00 (m, 4H), 1.82-1.66 (m,4H), 1.53-1.40 (m, 4H), 1.40-1.25 (m, 4H); HRMS (ESI-MS m/z) calculatedfor C₂₆H₄₆N₁₀O₄S₂ [M+H]⁺, 627.3223. found 627.3238

31. Preparation of EE:(R)-2-amino-N-(2-(1,4-dihydrobenzo[d][1,2]dithiin-6-yloxy)ethyl)-6-guanidinohexanamide

A solution of 9 (300 mg, 0.38 mmol) in a mixture of THF (15 mL),methanol (15 mL), and water (15 mL) was charged with solid LiOH.H₂O (64mg, 1.53 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture was charged with TCEP.HCl (54 mg,0.19 mmol) and stirred for another 1 h. The organic solvent was removedand the residue was partitioned between saturated aqueous NaHCO₃solution (10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated andthe aqueous layer was extracted with CH₂Cl₂ (2×30 mL). The combinedorganic layers were dried over Na₂SO₄, filtered, and concentrated. Thefinal residue was dissolved in EtOH (15 mL) and 4 N HCl (15 mL) wasadded. After stirring at room temperature for 1 h, the reaction mixturewas concentrated to afford compound B (155 mg, crude HCl salt) as ayellow solid. A solution of EE (155 mg, 0.38 mmol, crude) in methanol(10 mL), was charged with NH₄OH (30% in water, 10 mL) and DMSO (0.5 mL)and reaction mixture was stirred at room temperature for 20 h in openair (>90% formation of disulfide was observed by LCMS analyses). Solventwas removed, purified by reverse-phase column chromatography andlyophilized to afford 78 mg (52%, over two steps) of pure compoundP-2064 as a hygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.03 (d,J=8.4 Hz, 1H), 6.78 (dd, J=8.4, 2.6 Hz, 1H), 6.72 (d, J=2.6 Hz, 1H),4.07 (ddd, J=6.1, 3.5, 1.6 Hz, 2H), 4.03 (s, 2H), 4.00 (s, 2H), 3.85 (t,J=6.8 Hz, 1H), 3.70 (td, J=14.4, 4.6 Hz, 1H), 3.55 (ddd, J=14.4, 6.6,4.7 Hz, 1H), 3.05 (t, J=7.4 Hz, 2H), 1.92-1.78 (m, 2H), 1.65-1.52 (m,2H), 1.50-1.36 (m, 2H); ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (t, J=5.0 Hz,1H), 8.21 (br s, 3H), 7.82 (br s, 1H), 7.59-6.70 (m, 7H), 7.08 (d, J=8.5Hz, 1H), 6.81 (dd, J=8.5, 2.7 Hz, 1H), 6.78 (d, J=2.7 Hz, 1H), 4.08 (s,2H), 4.05 (s, 2H), 4.04-3.98 (m, 2H), 3.75 (t, J=6.8 Hz, 1H), 3.58-3.42(m, 2H), 3.03 (dd, J=13.0, 6.3 Hz, 2H), 1.76-1.64 (m, 2H), 1.54-1.39 (m,2H), 1.38-1.27 (m, 2H); HRMS (ESI-MS m/z) calculated for C₁₇H₂₇N₅O₂S₂[M+H]⁺, 398.1684. found 398.1674.

32. Preparation of FF:(S,2S,2′S)-N,N′-(2,2′-(1,4-dihydrobenzo[d][1,2]dithiine-6,7-diyl)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2,6-diaminohexanamido)hexanamide)

A solution of X (50 mg, 0.05 mmol) in methanol (10 mL), was charged withNH₄OH (30% in water, 10 mL) and DMSO (0.5 mL) and reaction mixture wasstirred at room temperature for 16 h in open air (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 76 mg(49%)* of pure compound FF as a hygroscopic white solid: ¹H NMR (400MHz, CD₃OD) δ 6.79 (s, 2H), 4.10 (t, J=5.5 Hz, 4H), 3.98 (s, 4H), 3.97(t, J=6.6 Hz, 2H), 3.91 (t, J=7.1 Hz, 2H), 3.71 (td, J=14.1, 5.5 Hz,2H), 3.59 (td, J=14.2, 5.1 Hz, 2H), 3.27-3.17 (m, 2H), 3.15-3.07 (m,2H), 2.96 (dd, J=8.5, 7.6 Hz, 4H), 1.99-1.78 (m, 8H), 1.77-1.68 (m, 4H),1.61-1.40 (m, 12H); ¹H NMR (400 MHz, DMSO-d₆) δ 9.04 (t, J=5.1 Hz, 2H),8.75 (t, J=5.4 Hz, 2H), 8.23 (br s, 12H), 6.83 (s, 2H), 4.02 (s, 4H),4.01 (t, J=6.6 Hz, 4H), 3.85 (t, J=6.6 Hz, 2H), 3.77 (t, J=6.3 Hz, 2H),3.58-3.40 (m, 4H), 3.05 (dd, J=11.6, 6.2 Hz, 4H), 2.75 (t, J=7.5 Hz,4H), 1.86-1.66 (m, 8H), 1.65-1.51 (m, 4H), 1.50-1.27 (m, 12H); HRMS(ESI-MS m/z) calculated for C₃₆H₆₆N₁₀O₆S₂ [M+H]⁺, 799.4686; found799.4726.

33. Preparation of GG:(S,2S,2′S)-N,N′-(2,2′-(1,4-dihydrobenzo[d][1,2]dithiine-6,7-diyl)bis(oxy)bis(ethane-2,1-diyl))bis(2-amino-6-((S)-2-amino-6-guanidinohexanamido)hexanamide)

A solution of Y (60 mg, 0.054 mmol) in methanol (10 mL), was chargedwith NH₄OH (30% in water, 10 mL) and DMSO (0.5 mL) and reaction mixturewas stirred at room temperature for 20 h in open air (>90% formation ofdisulfide was observed by LCMS analyses). Solvent was removed, purifiedby reverse-phase column chromatography and lyophilized to afford 42 mg(33%)* of pure compound GG as a hygroscopic white solid: NMR (400 MHz,CD₃OD) δ 6.79 (s, 2H), 4.10 (t, J=5.6 Hz, 4H), 3.98 (s, 4H), 3.97 (t,J=6.7 Hz, 2H), 3.89 (t, J=6.6 Hz, 2H), 3.71 (td, J=14.3, 5.7 Hz, 2H),3.59 (td, J=14.1, 5.1 Hz, 2H), 3.22 (t, J=6.9 Hz, 4H), 3.19-3.08 (m,4H), 1.98-1.76 (m, 8H), 1.73-1.60 (m, 4H), 1.60-1.38 (m, 12H); ¹H NMR(400 MHz, DMSO-d₆) δ 9.03 (t, J=5.4 Hz, 2H), 8.73 (t, J=5.4 Hz, 2H),8.40-8.18 (m, 12H), 7.85 (t, J=5.5 Hz, 2H), 7.69-6.83 (m, 7H), 6.83 (s,2H), 4.02 (s, 4H), 4.01 (t, J=6.2 Hz, 2H), 3.90-3.81 (m, 2H), 3.80-3.71(m, 2H), 3.58-3.39 (m, 4H), 3.15-3.01 (m, 8H), 1.85-1.64 (m, 8H),1.58-1.27 (m, 16H); HRMS (ESI-MS m/z) calculated for C₃₈H₇₀N₁₄O₆S₂[M+H]⁺, 883.5122; found 883.5134.

* 100 mg of crude P-2061 (obtained after combining impure fractions ofSG-SJL-D-87) was also subjected to similar reaction condition for cyclicdisulfide formation; after purification by reverse phase column combinedwith above for lyophilization. * Yield is based on combined startingmaterials (60 mg+100 mg).

34. Preparation of HH:(R)-2-amino-N-((R)-5-amino-6-(2-(1,4-dihydrobenzo[d][1,2]dithiin-6-yloxy)ethylamino)-6-oxohexyl)-6-guanidinohexanamide

A solution of 17 (1.30 g, 1.28 mmol) in a mixture of THF (20 mL),methanol (20 mL), and water (20 mL) was charged with solid LiOH.H₂O (216mg, 5.14 mmol) and the reaction mixture was stirred at room temperaturefor 1 h. The above reaction mixture TCEP.HCl (183 mg, 0.64 mmol) wascharged with and stirred for another 1 h. The solvent was removed andthe residue was partitioned between saturated aqueous NaHCO₃ solution(10 mL) and CH₂Cl₂ (50 mL). The CH₂Cl₂ layer was separated and theaqueous layer was extracted with CH₂Cl₂ (2×30 mL). The organic layerswere combined, dried over Na₂SO₄, filtered, and concentrated. Theresidue was dissolved in EtOH (20 mL) and 4 N HCl (20 mL) was added.After stirring at room temperature for 1 h, the reaction mixture wasconcentrated to afford compound D (820 mg, crude HCl salt) as a yellowsolid. A solution of D (220 mg, 0.34 mmol, crude) in methanol (10 mL),was charged with NH₄OH (30% in water, 10 mL) and DMSO (0.1 mL) andreaction mixture was stirred at room temperature for 20 h in open air(>90% formation of disulfide was observed by LCMS analyses). Solvent wasremoved, purified by reverse-phase column chromatography and lyophilizedto afford 30 mg (17%, over two steps) of pure compound P-2067 as ahygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.04 (d, J=8.4 Hz,1H), 6.82 (dd, J=8.4, 2.6 Hz, 1H), 6.78 (d, J=2.6 Hz, 1H), 4.10-4.05 (m,2H), 4.03 (s, 2H), 4.00 (s, 2H), 3.90 (dt, J=6.8, 1.4 Hz, 2H), 3.75-3.67(m, 1H), 3.59-3.51 (m, 1H), 3.31 (t, J=7.4 Hz, 2H), 3.18-3.06 m, 2H),1.97-1.72 (m, 4H), 1.71-1.58 (m, 2H), 1.57-1.38 (m, 6H); ¹H NMR (400MHz, DMSO-d₆) δ 8.82 (t, J=5.5 Hz, 1H), 8.66 (t, J=5.4 Hz, 1H), 8.25 (brs, 6H), 7.80 (t, J=5.2 Hz, 1H), 7.66-6.67 (m, 6H), 7.08 (d, J=8.5 Hz,1H), 6.81 (dd, J=8.5, 2.7 Hz, 1H), 6.76 (d, J=2.7 Hz, 1H), 4.08 (s, 2H),4.05 (s, 2H), 4.04-3.98 (m, 2H), 3.80-3.69 (m, 2H), 3.62-3.40 (m, 3H),3.15-2.99 (m, 4H), 1.79-1.65 (m, 4H), 1.53-1.35 (m, 4H), 1.37-1.27 (m,4H); ESI-MS (m/z) C₂₃H₃₉N₇O₃S₂+H⁺526.

35. Preparation JJ:(2R,2′R)-N,N′-(3,3′-(2-(1,4-dihydrobenzo[d][1,2]dithiin-6-yloxy)ethylazanediyl)bis(propane-3,1-diyl))bis(2-amino-6-guanidinohexanamide)

A solution of 57 (475 mg, 0.35 mmol, mixture) in a mixture of THF (15mL), methanol (15 mL), and water (15 mL) was charged with solid LiOH.H₂O(59 mg, 1.40 mmol) and the reaction mixture was stirred at roomtemperature for 1 h. The above reaction mixture was charged withTCEP.HCl (50 mg, 0.17 mmol) and stirred for another 1 h. The solvent wasremoved and the residue was partitioned between CH₂Cl₂ (50 mL) andsaturated aqueous NaHCO₃ solution (10 mL). The CH₂Cl₂ layer wasseparated and the aqueous layer was extracted with CH₂Cl₂ (2×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered, andconcentrated. The residue was dissolved in EtOH (15 mL) and 4 N HCl (15mL) was added. After stirring at room temperature for 1 h, the reactionmixture was concentrated to afford compound Z (300 mg, crude HCl salt)as a yellow solid. (200 mg, 0.24 mmol, crude) in methanol (10 mL), wascharged with NH₄OH (30% in water, 10 mL) and DMSO (0.1 mL) and reactionmixture was stirred at room temperature for 20 h in open air (>90%formation of disulfide was observed by LCMS analyses). Solvent wasremoved, purified by reverse-phase column chromatography and lyophilizedto afford 80 mg (39%, over two steps) of pure compound JJ as ahygroscopic white solid: ¹H NMR (400 MHz, CD₃OD) δ 7.09 (d, J=8.4 Hz,1H), 6.89 (dd, J=8.4, 2.6 Hz, 1H), 6.87 (d, J=2.6 Hz, 1H), 4.40 (t,J=4.8 Hz, 2H), 4.07 (s, 2H), 4.02 (s, 2H), 3.92 (t, J=6.6 Hz, 2H),3.72-3.66 (m, 2H), 3.51-3.32 (m, 8H), 3.21 (dt, J=7.2, 1.9 Hz, 4H),2.19-2.02 (m, 4H), 2.00-1.77 (m, 4H), 1.70-1.57 (m, 4H), 1.54-1.42 (m,4H); ¹H NMR (400 MHz, DMSO-d₆) δ 10.83 (br s, 1H), 8.96 (dd, J=102, 5.8Hz, 2H), 8.37 (br s, 6H), 7.85 (t, J=5.5 Hz, 2H), 7.67-6.78 (m, 7H),7.11 (d, J=8.6 Hz, 1H), 6.89 (dd, J=8.6, 2.4 Hz, 1H), 6.88 (d, J=2.4 Hz,1H), 4.40 (t, J=4.6 Hz, 2H), 4.11 (s, 2H), 4.07 (s, 2H), 3.82-3.72 (m,2H), 3.58-3.50 (m, 2H), 3.31-3.17 (m, 8H), 3.15-3.03 (m, 4H), 2.06-1.87(m, 4H), 1.79-1.66 (m, 4H), 1.54-1.41 (m, 4H), 1.39-1.26 (m, 4H); HRMS(ESI-MS m/z) calculated for C₃₀H₅₅N₁₁O₃S₂ [M+H]⁺, 682.4009. found682.404

36. Preparation of (4-(2-aminoethoxy)-1,2-phenylene)dimethanethiol (KK)

37. Preparation ofLL:1-(2-(3,4-bis(mercaptomethyl)phenoxy)ethyl)guanidine (LL)

All of the references cited above throughout this application areincorporated herein by reference. In the event of a conflict between theforegoing description and a reference, the description provided hereincontrols.

That which is claimed is:
 1. A compound represented by formula Ia, Ib,Ic or Id:

wherein R¹ and R² are each, independently, hydrogen, lower alkyl,halogen or triflouromethyl; R³ and R⁴ are each, independently, hydrogen,lower alkyl, hydroxyl-lower alkyl, phenyl, (phenyl)-lower alkyl,(halophenyl)-lower alkyl, ((lower-alkyl)phenyl)-lower-alkyl,((lower-alkoxy)phenyl)-lower-alkyl, (naphthyl)-lower-alkyl, or(pyridyl)-lower-alkyl; each R⁵ is, independently, hydrogen, halogen,trifluoromethyl, lower alkyl, unsubstituted or substituted phenyl, loweralkyl-thio, phenyl-lower alkyl-thio, lower alkyl-sulfonyl, orphenyl-lower alkyl-sulfonyl, OH, —(CH₂)_(m)—OR⁸, —O—(CH₂)_(m)—OR⁸,—(CH₂)_(n)—NR⁷R¹⁰, —(CH₂)_(n)—NR⁷R⁷, —O—(CH₂)_(m)—NR⁷R¹⁰,—O—(CH₂)_(m)—NR⁷R⁷, —(CH₂)—(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂CH₂O)_(m)—R⁸,—O—(CH₂CH₂O)_(m)—R⁸, —(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰,—O—(CH₂CH₂O)_(m)—CH₂CH₂NR⁷R¹⁰, —(CH₂)_(n)—C(═O)NR⁷R¹⁰,—O—(CH₂)_(m)—C(═O)NR⁷R¹⁰, —(CH₂)_(n)—(Z)_(g)—R⁷,—O—(CH₂)_(m)—(Z)_(g)—R⁷, —(CH₂)_(n)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸,—O—(CH₂)_(m)—NR¹⁰—CH₂(CHOR⁸)(CHOR⁸)_(n)—CH₂OR⁸, —(CH₂)_(n)—CO₂R⁷,—O—(CH₂)_(m)—CO₂R⁷, —OSO₃H, —O-glucuronide, —O-glucose,

-Link-(CH₂)_(m)—CAP, -Link-(CH₂)_(n)(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂CH₂O)_(m)—CH₂—CAP, -Link-(CH₂CH₂O)_(m)—CH₂CH₂—CAP,-Link-(CH₂)_(m)—(Z)_(g)-CAP, -Link-(CH₂)_(n)(Z)_(g)—(CH₂)_(m)-CAP,-Link-(CH₂)_(n)—NR¹³—CH₂(CHOR⁸)(CHOR⁸)_(n)-CAP,-Link-(CH₂)_(n)—(CHOR⁸)_(m)CH₂—NR¹³—(Z)_(g)-CAP,-Link-(CH₂)_(n)NR¹³—(CH₂)_(m)(CHOR⁸)_(n)CH₂NR¹³—(Z)_(g)-CAP, -Link-(CH₂)_(m)—(Z)_(g)—(CH₂)_(m)-CAP, -Link-NH—C(═O)—NH—(CH₂)_(m)-CAP,-Link-(CH₂)_(m)—C(═O)NR¹³—(CH₂)_(m)—CAP,-Link-(CH₂)_(n)—(Z)_(g)—(CH₂)_(m)—(Z)_(g)-CAP, or-Link-Z_(g)—(CH₂)_(m)-Het-(CH₂)_(m)—CAP with the proviso that at leastone R⁵ group contains at least one basic nitrogen; each R⁷ is,independently, hydrogen, lower alkyl, phenyl, substituted phenyl, loweralkyl phenyl or —CH₂(CHOR⁸)_(m)—CH₂OR⁸; each R⁸ is, independently,hydrogen, lower alkyl, lower alkyl phenyl, —C(═O)—R¹¹, glucuronide,2-tetrahydropyranyl, or

each R⁹ is, independently, —CO₂R⁷, —CON(R⁷)₂, —SO₂CH₃, —C(═O)R⁷,—CO₂R¹³, —CON(R¹³)₂, —SO₂CH₂R¹³, or —C(═O)R¹³; each R¹⁰ is,independently, —H, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷, or—CH₂—(CHOH)_(n)—CH₂OH; each Z is, independently, —(CHOH)—, —C(═O)—,—(CHNR⁷R¹⁹)—, —(C═NR¹⁹)—, —NR¹⁹—, —(CH₂)_(n)—, —(CHNR¹³R¹³)—,—(C═NR¹³)—, or —NR¹³—; each R¹¹ is, independently, hydrogen, loweralkyl, phenyl lower alkyl or substituted phenyl lower alkyl; each R¹²is, independently, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,—CH₂(CHOH)_(n)—CH₂OH, —CO₂R¹³, —C(═O)NR¹³R¹³, or —C(═O)R¹³; each R¹³ is,independently, hydrogen, lower alkyl, phenyl, substituted phenyl or—CH₂(CHOR⁸)_(m)—CH₂OR⁸, —SO₂CH₃, —CO₂R⁷, —C(═O)NR⁷R⁹, —C(═O)R⁷,—CH₂—(CHOH)_(n)—CH₂OH, —(CH₂)_(m)—NR⁷R¹⁰, —(CH₂)_(m)—NR⁷R⁷,—(CH₂)_(m)NR¹¹R¹¹, —(CH₂)_(m)(NR¹¹R¹¹R¹¹)⁺,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR¹¹R¹¹,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R¹⁰, —(CH₂)_(m)—NR¹⁰R¹⁰,—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)—(NR¹¹R¹¹R¹¹)⁺, or—(CH₂)_(m)—(CHOR⁸)_(m)—(CH₂)_(m)NR⁷R⁷; each g is, independently, aninteger from 1 to 6; each m is, independently, an integer from 1 to 7;each n is, independently, an integer from 0 to 7; each -Het- is,independently, —N(R⁷)—, —N(R¹⁰)—, —S—, —SO—, —SO₂—; —O—, -sO₂NH—,—NHSO₂—, —NR⁷CO—, —CONR⁷—, —N(R¹³)—, —SO₂NR¹³—, —NR¹³CO—, or —CONR¹³—;each Link is, independently, —O—, —(CH₂)_(n)—, —O(CH₂)_(m)—,—NR¹³—C(═O)—NR¹³—, —NR¹³—C(═O)—(CH₂)_(m)—, —C(═O)NR¹³—(CH₂)_(m) ⁻,—(CH₂)_(n)—(Z)_(g)—(CH₂)_(n)—, —S—, —SO—, —SO₂—, —SO₂NR⁷—, —SO₂NR¹⁰—, or-Het-; each CAP is, independently

with the proviso that when any —CHOR⁸— or —CH₂OR⁸ groups are located1,2- or 1,3- with respect to each other, the R⁸ groups may, optionally,be taken together to form a cyclic mono- or di-substituted 1,3-dioxaneor 1,3-dioxolane; and racemates, enantiomers, diastereomers, tautomers,polymorphs, pseudopolymorphs and pharmaceutically acceptable salts,thereof.
 2. A method of liquefying mucus from mucosal surfaces,comprising: administering an effective amount of the compound of claim 1to a mucosal surface of a subject.
 3. A method of treating chronicbronchitis, treating bronchiectasis, treating cystic fibrosis, treatingchronic obstructive pulmonary disease, treating asthma, treatingsinusitis, treating vaginal dryness, treating dry eye, promoting ocularhydration, promoting corneal hydration, promoting mucus clearance inmucosal surfaces, treating Sjogren's disease, treating distal intestinalobstruction syndrome, treating dry skin, treating esophagitis, treatingdry mouth, treating nasal dehydration, treating ventilator-inducedpneumonia, treating asthma, treating primary ciliary dyskinesia,treating otitis media, inducing sputum for diagnostic purposes, treatingcystinosis, treating emphysema, treating pneumonia, treatingconstipation, treating chronic diverticulitis, and/or treatingrhinosinusitis, comprising: administering an effective amount of thecompound of claim 1 to a subject in need thereof.
 4. A method oftreating an eye disease characterized by the presence of oculardischarge consisting of administering to a subject in need an effectiveamount of the compound of claim
 1. 5. The method of claim 4, wherein theeye disease is one or more conditions selected from the group consistingof blepharitis, allergies, conjunctivitis, corneal ulcer, trachoma,congenital herpes simplex, corneal abrasions, ectropion, eyeliddisorders, gonococcal conjunctivitis, herpetic keratitis, ophthalmitis,Sjogren's Syndrome, or Stevens-Johnson Syndrome.
 6. A method of treatinga disease ameliorated by increased mucociliary clearance and mucosalhydration comprising administering to a subject in need of increasedmucociliary clearance and mucosal hydration an effective amount of anosmolyte and the compound of claim
 1. 7. The method of claim 6, whereinthe disease is one or more conditions selected from the group consistingof chronic bronchitis, bronchiectasis, cystic fibrosis, asthma,sinusitis, vaginal dryness, dry eye, Sjogren's disease, distalintestinal obstruction syndrome, dry skin, esophagitis, dry mouth(xerostomia), nasal dehydration, asthma, primary ciliary dyskinesia,otitis media, chronic obstructive pulmonary disease, emphysema,pneumonia, diverticulitis, rhinosinusitis, and airborne infections. 8.The method of claim 6, wherein the compound is administered precedingadministration of the osmolyte.
 9. The method of claim 6, wherein thecompound is administered concurrent with administration of the osmolyte.10. The method of claim 6, wherein the compound is administeredfollowing administration of the osmolyte.
 11. The method of claim 6,wherein the osmolyte is hypertonic saline or mannitol.
 12. The method ofclaim 6, wherein the osmolyte is sodium chloride which is delivered as amicronized particle of respirable size.
 13. The method of claim 6,wherein the effective amount of an osmolyte and a compound of Formula(I) is administered by aerosolization using a device capable ofdelivering the formulation to the nasal passages or pulmonary airwaywherein the aerosol is a respirable size.
 14. A composition, comprising:(a) the compound of claim 1 and (b) an osmotically active compound. 15.A method of inducing sputum, comprising administering to a subject inneed of increased mucociliary clearance and mucosal hydration aneffective amount of an osmolyte and the compound of claim
 1. 16. Amethod of prophylactic, post-exposure prophylactic, preventive ortherapeutic treatment against diseases or conditions caused bypathogens, comprising administering to a subject in need of increasedmucociliary clearance and mucosal hydration an effective amount of thecompound of claim
 1. 17. The method of claim 16, wherein the pathogen isanthrax or plague.
 18. A method for preventing, mitigating, and/ortreating deterministic health effects to the respiratory tract and/orother bodily organs caused by respirable aerosols containingradionuclides in a human in need thereof, said method comprisingadministering to said human an effective amount of the compound of claim1, or a pharmaceutically acceptable salt thereof.
 19. A pharmaceuticalcomposition, comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier.
 20. A method for improving mucus penetration oftherapeutic agents comprising administering an effective amount of thecompound of claim 1 and a second therapeutic agent.
 21. The method ofclaim 20, wherein the therapeutic agents is an osmolyte, a sodiumchannel blocker, a secretogogue, a bronchodilator, an anti-infective, ananti-inflammatory, or a gene carrier.
 22. A method for decreasingmucosal inflammation comprising administering an effective amount of thecompound of claim
 1. 23. A method for decreasing mucosal oxygen freeradicals comprising administering an effective amount of the compound ofclaim
 1. 24. The compound of claim 1, which is one of the followingcompounds:


25. The compound of claim 1, which is an acid addition salt of aninorganic acid or an organic acid selected from the group consisting ofhydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid,polyglutamic acid, naphthalensulfonic acid, methanesulfonic acid,p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonicacid, malonic acid, sulfosalicylic acid, glycolic acid,2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalicacid, mandelic acid, and lactic acid.